Transgenerational epigenetics and brain disorders.

CHAPTER TWO

Transgenerational Epigenetics and Brain Disorders Nadia Rachdaoui, Dipak K. Sarkar1 Rutgers Endocrine Research Program, Department of Animal Sciences, Rutgers University, New Brunswick, New Jersey, USA 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction to Transgenerational Epigenetic Inheritance 2. Epigenetics and Epigenetic Processes 3. Evidence for Transgenerational Epigenetic Inheritance 4. Germline-Independent Epigenetic Inheritance 5. Germline-Dependent Epigenetic Transmission 6. Transgenerational Epigenetic Effects on Brain Disorders 7. Concluding Remarks Acknowledgment References

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Abstract Neurobehavioral and psychiatric disorders are complex diseases with a strong heritable component; however, to date, genome-wide association studies failed to identify the genetic loci involved in the etiology of these brain disorders. Recently, transgenerational epigenetic inheritance has emerged as an important factor playing a pivotal role in the inheritance of brain disorders. This field of research provides evidence that environmentally induced epigenetic changes in the germline during embryonic development can be transmitted for multiple generations and may contribute to the etiology of brain disease heritability. In this review, we discuss some of the most recent findings on transgenerational epigenetic inheritance. We particularly discuss the findings on the epigenetic mechanisms involved in the heritability of alcohol-induced neurobehavioral disorders such as fetal alcohol spectrum disorders.

1. INTRODUCTION TO TRANSGENERATIONAL EPIGENETIC INHERITANCE Most complex human diseases such as cancer and psychiatric disorders are governed by a genetic heritable component; however, to date, International Review of Neurobiology, Volume 115 ISSN 0074-7742 http://dx.doi.org/10.1016/B978-0-12-801311-3.00002-0

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2014 Elsevier Inc. All rights reserved.

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Nadia Rachdaoui and Dipak K. Sarkar

genome-wide association studies failed to identify the causal loci and genetic basis of most complex diseases (Gibson, 2011). This “missing heritability” suggests that, in addition to genetically inherited information through particular loci, additional layers of information referred to as epigenetics play an important role in the inheritance of complex diseases (Bohacek & Mansuy, 2013; Danchin et al., 2011). Acquired epigenetic marks are thought to be completely erased between generations; however, several studies have shown that this epigenetic information can be transmitted through the germline. This phenomenon is known as “transgenerational epigenetic inheritance” (Daxinger & Whitelaw, 2010; Horsthemke, 2007). Moreover, the discovery of parental imprinting also called “genomic imprinting” in the 1980s provided the first evidence that epigenetic processes persist between generations and might underlie the transgenerational epigenetic inheritance of traits and diseases (Kearns, Preis, McDonald, Morris, & Whitelaw, 2000; Reik, Collick, Norris, Barton, & Surani, 1987; Swain, Stewart, & Leder, 1987; Tost, 2009). Genomic imprinting is a non-Mendelian form of gene regulation that contributes to the establishment of epigenetic marks in the parental gametes (Reik & Walter, 2001). The mechanisms for gene imprinting are still not fully elucidated; however, it is believed that they involve epigenetic silencing through methylation of CpG-rich domains in a parent-specific manner during gametonenesis (Pfeifer, 2000). This phenomenon results in the preferential expression of specific genes from the allele inherited either from the father or from the mother. For example, the imprinted gene insulin-like growth factor 2 (Igf2), which was shown to play an important role in fetal development and growth, is exclusively expressed from the paternal allele; the maternally inherited allele for Igf2 is epigenetically silenced (Chao & D’Amore, 2008). Because of these epigenetically mediated allele-specific gene expressions, imprinted genes are believed to be especially susceptible to epigenetic dysregulation by environmental factors, such as nutrition, stress, and toxic agents. For example, it was shown that maternal exposure to methyldeficient diets during pregnancy can alter the expression of imprinted genes (Bekdash, Zhang, & Sarkar, 2013; Waterland, Lin, Smith, & Jirtle, 2006). When these imprinting aberrations occur during early fetal development, they are often manifested as developmental and neurological disorders. Evidence shows that among the different organs, the brain is the most enriched tissue in imprinted genes (Prickett & Oakey, 2012) and, therefore, the most vulnerable to environmental perturbations ( Jirtle & Skinner, 2007). Several research studies have reported that early-life exposure to environmental

Transgenerational Effects on Brain Function

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factors such as stress, drugs, and toxins can alter the epigenetic status of imprinted genes and other genes important to brain development and result in neurobehavioral deficiencies and psychiatric disorders (Jirtle & Skinner, 2007; Prickett & Oakey, 2012). Furthermore, it is suggested that certain inherited brain disorders such as Beckwith–Wiedemann syndrome, Rett syndrome, fragile X syndrome, and Angelman’s syndrome arise from abnormal-specific imprinted genes (Kaminsky, Wang, & Petronis, 2006; Kantor, Shemer, & Razin, 2006; Weksberg, Shuman, & Smith, 2005). In this review, we describe the most recent findings on transgenerational epigenetic inheritance, particularly in relation to brain disorders. We first discuss the mounting evidence that supports the transgenerational inheritance of environmentally induced epigenetic alterations and then we describe the epigenetic mechanisms involved in the alcohol-mediated neurobehavioral and cognitive deficiencies and their role in the transgenerational transmission of alcohol’s deleterious effects on brain development and function. We conclude this review by arguing that understanding the implications of these environmentally induced transgenerational epigenetic changes will extend our knowledge on human disease susceptibility and ultimately lead to the development of new diagnostic and therapeutic strategies.

2. EPIGENETICS AND EPIGENETIC PROCESSES Richard Goldschmidt, an integrative biologist, believed that early developmental exposure to events has as much impact as genetics on the determination of the adult phenotype (Goldschmidt, 1933). It is not until 1940 that the renowned embryologist Conrad Hal Waddington attempted to bridge both fields of genetics and embryology by being the first to coin the term “epigenetics.” Waddington described development as the path from genotype to phenotype and suggested that the mechanisms by which genes guide development or epigenetic, a process influenced by the surrounding environment, should be given the name of epigenetics. In his opinion, the epigenetic processes help to bridge the gap between environmental and genetic factors. In recent years, “epigenetics” is referred to as the study of mechanisms involved in changes in genetic information and gene expression that are independent of any change in DNA sequence (i.e., mutations). This process serves to maintain different gene expression patterns during key developmental periods and contributes to the determination of different cell

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phenotypes. It also constitutes a dynamic process that helps translate environmental stimuli into changes in gene expression patterns ( Jang & Serra, 2014; Jirtle et al., 2007; Reul, 2014). Many epigenetic processes have been identified. We look at some of the epigenetic marks that consist of DNA methylation at the carbon-5 position of cytosine on CpG dinucleotides, histone proteins posttranslational modifications (HPTMs) at their N-terminal tails by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, and sumoylation and interference of gene transcription by small noncoding RNAs (sncRNAs; Kugel & Goodrich, 2012). Epigenetic marks induce changes in chromatin structure and serve as docking sites for transcription factors (i.e., activators and repressors). They are specific to each gene and are dynamically regulated by various environmental conditions. In terms of gene activity, a condensed chromatin (heterochromatin) is generally repressed, whereas an open chromatin (euchromatin) is transcriptionally active and tends to be associated with distinct epigenetic signals. Heterochromatin is often associated with methylation of CpG dinucleotides, hypoacetylation of H3 and H4, and dimethylation/trimethylation of lysine 9 on H3 (H3K9Me2,3), whereas euchromatin is associated with hypomethylation of CpG dinucleotides, acetylation of H3 and H4, and dimethylation/trimethylation of lysine 4 on H3 (H3K4Me; Shukla et al., 2008). Another mechanism involved in modulating chromatin structure is the incorporation of nonallelic histone variants of H2A, H2B, and H3, and not H4, which replace preexisting conventional histones during development and differentiation (Bosch & Suau, 1995; Brandt et al., 1979; Margueron & Reinberg, 2010; Wunsch, Reinhardt, & Lough, 1991). This selective deposition of histone variants may become predominant in the differentiated cell (Pina & Suau, 1987a, 1987b; Wunsch et al., 1991). Several enzymes are involved in these epigenetic processes. Histone acetyltransferases acetylate lysine residues on the N-terminal tail of histone proteins, decreasing its affinity for DNA and resulting in the relaxation of chromatin making it accessible to the transcription machinery. In contrast, histone deacetylases remove the acetyl groups and cause chromatin condensation and gene repression (Van Holde & Zlatanova, 1996). Two families of DNA methyltransferases (Dnmts) have been identified, maintenance and de novo methyltransferases (Bestor, 2000). DNA methylation states are mitotically maintained through the action of DNMT1, a maintenance methyltransferase that acts primarily on hemimethylated DNA (Yoder, Soman,


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Verdine, & Bestor, 1997). In contrast, the novo methyltransferases such as DNMT3A and DNMT3B act on unmethylated DNA and their activity is crucial during early developmental stages when DNA methylation patterns are being established (La Salle et al., 2004; Yokochi & Robertson, 2002). The activity of DNMT1, however, is more crucial to maintaining these methylation patterns through adulthood (Yoder et al., 1997). Recent evidence has demonstrated that the mammalian transcriptome is extremely complex, and that it includes a number of sncRNAs (20–30 nucleotides), such as short-interfering RNAs, microRNAs (miRNAs), and PIWI-interacting RNAs (Aalto & Pasquinelli, 2012), which have been implicated in epigenetic silencing of specific genes and in the protection of the genome against viruses and transposons (Luteijn & Ketting, 2013; Moazed, 2009). Epigenetic changes can occur in any cell of the body, but when they occur in the germ cells, they can be passed on to the next generation. While they are required for normal development, they can also contribute to disease onset. Disruption of any of these epigenetic processes can cause faulty activation or silencing of genes and be associated with risks of complex diseases such as cancer, diabetes, and brain disorders.

3. EVIDENCE FOR TRANSGENERATIONAL EPIGENETIC INHERITANCE Transgenerational epigenetic inheritance is the heritable or familial transmission of environmentally induced epigenetic variations that last over multiple generations. It is important to differentiate between the transgenerational effects, which are due to a single exposure to an environmental insult and which are transmitted, through the germline, for multiple generations; and the multigenerational effects which are due to the continuous exposure of multiple consecutive generations to the same environmental factor (Skinner & Guerrero-Bosagna, 2009). A transgenerational epigenetic basis of environmental effects can be considered only if they are passed on for at least three generations for maternal exposure and two generations for paternal exposure (summarized in Fig. 2.1; Jirtle et al., 2007). We will name these two routes of transmission, germline-dependant and germline-independent inheritance, to distinguish between the transgenerational effects and the multigenerational effects of environmentally induced epigenetic variations (Fig. 2.1).

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Germline-independent inheritance

Germline-dependent inheritance

Environmental insult

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Life experiences and social interactions (e.g., Maternal care)

B Exposed female

F0

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Transgenerational epigenetic inheritance

F1

Exposed gametes

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F2

Transgenerational epigenetic inheritance

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F0 Exposed F1 fetus and F2 gametes

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F0 mother

Exposed male

Somatic epigenetic modifications in F1 offspring

F1 mother F1 F2


F2 mother F2 F3


F3

Figure 2.1 Germline-dependent versus germline-independent epigenetic inheritance. Different routes for the transmission of epigenetic modifications across multiple generations have been proposed. The present scheme summarizes the different paths of epigenetic inheritance. In the germline-dependent route of inheritance, two mechanisms have been suggested: (A) exposure of a gestating mother (F0) to an environmental stressor leads to the direct exposure of three consecutive generations to the same environmental factor, the mother (F0), the fetus (F1), and the F1 germline from which originates the F2 generation. The transgenerational effect in this case is only observed at the F3 generation since the latter was never directly exposed to the environmental factor. (B) In the case of an F0 male exposure to an environmental factor, the transgenerational effect is seen at the F2 generation. One of the mechanisms implicated in this epigenetic inheritance involves epigenetic modifications in sperm cells (e.g., DNA methylation, HPTMs, and sncRNA interference). The other well-known mechanism of epigenetic


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4. GERMLINE-INDEPENDENT EPIGENETIC INHERITANCE Also referred to as “context dependent” is acquired through social and behavioral interactions and through parental and grandparental effects, as described by Weaver (2007) and others (McGowan et al., 2011) in their studies on the impact of maternal care during the early postnatal period. For example, good maternal care through grooming and licking was associated with a decrease in DNA methylation at the nerve growth factorinducible protein-A transcription factor binding site in the promoter region of the glucocorticoid receptor (GR) in the hippocampus of offspring rats (Weaver, 2007; Weaver et al., 2007). This effect was associated with stress resilience and was passed on to the next generation. In addition, early postnatal crossfostering of these pups with low licking and grooming mothers reversed these effects, which suggests that the transmission of these epigenetic changes is germline independent (Weaver, 2009).

5. GERMLINE-DEPENDENT EPIGENETIC TRANSMISSION This type of transgenerational transmission of epigenetic marks requires the action of environmental factors during germline differentiation, a critical narrow window during which the germline undergoes epigenetic reprogramming (Jirtle et al., 2007; Skinner, Manikkam, & GuerreroBosagna, 2010). The most sensitive developmental period to environmental perturbations is the fetal gonadal sex determination period (Anway, Cupp, Uzumcu, & Skinner, 2005; Skinner et al., 2010). After fertilization, the primordial germ cells undergo an erasure of DNA methylation to produce pluripotent embryonic stem cells. At the blastula stage of embryonic development, during gonadal sex determination, the DNA is remethylated in a sex-specific manner to generate male or female germlines (Morgan, Santos, Green, Dean, & Reik, 2005; Reik, Dean, & Walter, 2001). inheritance does not involve the transmission of epigenetic changes through the germline; the multigenerational transmission in this case is mediated through social interactions and early-life experiences. (C) For example, low maternal licking and grooming of pups, during the early postnatal period, lead to an increased DNA methylation of the promoter region of the GR and GR gene silencing. These epigenetic changes were associated with stress intolerance and were maintained in the adult female offspring (F1 mother) which in turn perpetuated the phenotype of low licking and grooming to the next generation of mothers (F2).

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However, it was suggested that during the final migration of primordial germ cells into the genital ridges, some germline imprinted genes or epimutations escape the DNA demethylation process allowing heritability of epigenetic changes across generations (Skinner et al., 2010). Following demethylation and differentiation of germ cells, new methylation patterns are reset and old patterns are maintained by the maintenance methyltransferase DNMT1, which recognizes DNA strands with old and de novo methylation patterns (Yoder et al., 1997). This maintenance process of imprinting is crucial to the appropriate expression of genes necessary for embryonic development but also to the transgenerational heritability of epigenetic changes (Skinner et al., 2010; Yoder et al., 1997). The first initial evidence for a transgenerational epigenetic inheritance through the germline came from the studies by Morgan, Sutherland, Martin, and Whitelaw (1999) and others of the inbred Agouti viable yellow (Avy) mouse strain. These Avy mice carry “transposable elements” in one allele of the agouti locus that can be epigenetically regulated through silencing by methylation. These alleles are referred to as metastable epialleles and in a hypomethylated state they are responsible for a yellow coat phenotype in Avy mice as compared to the agouti brown-colored coat of wild type. Intermediate levels of methylation, however, result in varying yellow patches of fur in these animals (Rosenfeld, 2010; Wolff, Kodell, Moore, & Cooney, 1998). In addition, alterations in HPTMs were also observed in these differentially methylated metastable epialleles, suggesting that several epigenetic processes are involved in the Avy epiallele gene regulation (Dolinoy, Weinhouse, Jones, Rozek, & Jirtle, 2010). Moreover, these differentially methylated Avy metastable epialleles can be transmitted to offspring through the maternal germline (Morgan et al., 1999). In another study by Blewitt, Vickaryous, Paldi, Koseki, and Whitelaw (2006), it was argued that DNA methylation is not the inherited epigenetic mark in Avy mice. These researchers found that, during the preimplantation phase immediately after fertilization, the paternally inherited allele is demethylated more rapidly than the maternal allele and that following maternal transmission there was no DNA methylation in the blastocyst. Moreover, when female mice carrying a Mel18 knockout allele (a mammalian polycomb group protein; Akasaka et al., 1996) were bred with Avy males, the haplo-insufficient Mel18 mice displayed epigenetic inheritance, whereas their wild-type littermates did not. These results suggest that events after the complete demethylation of the Avy allele are involved in this germline-dependent epigenetic inheritance and that DNA methylation is not the inherited epigenetic mark (Blewitt et al., 2006).


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Studies on the role of HPTMs in germline-dependent transgenerational epigenetic inheritance are still scarce. Studies on sperm cells have shown that 98% of HPTMs in mice and 85% in humans are lost when histones are replaced by protamines during spermatogenesis (Fischer, Sananbenesi, Mungenast, & Tsai, 2010; Hammoud et al., 2009; Johnson et al., 2011). The remaining histones play an important role in gene regulation during embryogenesis (Puri, Dhawan, & Mishra, 2010). In a study by Brykczynska et al. (2010), it was shown that the trimethylated lysine 27 on histone H3 (H3K27me3) mark was maintained in human sperm cells and that it plays an important role in spermatogenesis and in paternal transmission of epigenetic information (Brykczynska et al., 2010). Brunner, Nanni, and Mansuy (2014), using a novel proteomic approach, identified a total of 26 posttranslational modifications (PTMs) on specific residues of the core histones H2B, H3, and H4, and the linker histone H1 and 11 novel PTMs on the protamines PRM1 and PRM2 which clearly suggests the presence of epigenetic marks on histones and protamines in adult mouse sperm (Brunner et al., 2014). In our own work, we attempted to answer the question of whether alcohol-related disorders are transgenerationally inherited through multiple generations. We tested this hypothesis by using an animal model of fetal alcohol exposure. We found that ethanol exposure of pregnant dams leads to proopiomelanocortin (Pomc) gene expression deficiency in β-endorphin neurons of offspring, an effect that was due to Pomc gene epigenetic alterations. These effects of ethanol on the Pomc gene were associated with some of the pathologies observed in the fetal alcohol spectrum disorders (FASD) patients (Boyadjieva, Ortiguela, Arjona, Cheng, & Sarkar, 2009; Rachdaoui & Sarkar, 2013; Sarkar, Kuhn, Marano, Chen, & Boyadjieva, 2007), such as anxiety, stress hyperresponsiveness, immune deficiencies, and cancer susceptibility. More interestingly, these effects were maintained for over three generations (i.e., F1, F2, and F3) through the male germline and involved the transmission of epigenetic marks in sperm cells (Govorko, Bekdash, Zhang, & Sarkar, 2012). We will further discuss the data from these studies in Section 6. sncRNAs have also been detected in mature sperm and oocytes (Hamatani, 2012; Suh & Blelloch, 2011). Their role in the germ cells is still not clear; however, several recent studies have shown that sperm sncRNAs can be delivered to the oocyte during fertilization and contribute to the regulation of genes important for embryonic development (Liu et al., 2012; Ostermeier, Miller, Huntriss, Diamond, & Krawetz, 2004). It was also

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recently suggested that sncRNAs are involved in promoting transgenerational gene silencing and epigenetic inheritance across generations (Feng & Guang, 2013). For example, Kiani and colleagues showed that there were no differences in the DNA methylation pattern of the Kit locus (responsible for the fur color) between the wild-type and the mutant mice, where an RNA-mediated epigenetic modification of the Kit gene is transgenerationally transmitted in a non-Mendelian way. However, RNA bisulfite sequencing showed a Dnmt2-dependent tRNA methylation in sperm cells and a Dnmt2-dependent cytosine methylation in Kit RNA in mutant embryos. Taken together, these studies suggest an important role of Dnmt2 in sncRNA-mediated transgenerational epigenetic inheritance in this animal model (Kiani et al., 2013). The studies discussed herein clearly suggest that epigenetic marks persist in the germline and that they can be transmitted across generation. The mechanism by which the germline’s epigenetic material is transmitted to somatic cells is still unclear; however, it is believed that an altered germline epigenome, induced by environmental insults, can promote the transmission of abnormal states of cell and tissue differentiation and contribute to an increased risk of developing adult-onset diseases later in life, a theory referred to as “developmental origins of adult-onset disease” (Godfrey, Lillycrop, Burdge, Gluckman, & Hanson, 2007; Skinner, 2011). Studies are needed to identify the mechanisms that underlie this germline epigenetic transmission which may perhaps help in the understanding and prevention of numerous environmentally induced complex diseases.

6. TRANSGENERATIONAL EPIGENETIC EFFECTS ON BRAIN DISORDERS It is well established that during the prenatal and postnatal periods of brain development, there is an enhanced sensitivity to environmental factors. Importantly, these critical periods of brain development are characterized by extensive and rapid changes in neuronal and synaptic organization, processes that are associated with peak periods of epigenetic reprogramming. Epigenetic alterations, during brain development, by environmental factors such as stress, toxins, drugs, and traumatic events can have detrimental effects on brain function and result in neurobehavioral deficiencies and even psychiatric disorders (Tomalski & Johnson, 2010). For instance, in animal studies, it was shown that early-life exposure to stressors was associated with increased expression of genes involved in the hypothalamic–pituitary–adrenal


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response to stress and decreased expression of genes involved in suppressing that response (Ladd, Huot, Thrivikraman, Nemeroff, & Plotsky, 2004; Maccari et al., 2003). In addition, these environmentally induced developmental effects are transmitted across generations, leading to neurological and behavioral changes in offspring (Champagne, Francis, Mar, & Meaney, 2003; Franklin et al., 2010). In a mouse model combining maternal separation and maternal stress, it was reported that stressed pups (F1 generation) develop depressive behavior, altered response to novelty, deficits in risk assessment, and abnormal social behaviors (Franklin et al., 2010; Weiss, Franklin, Vizi, & Mansuy, 2011). In addition, both F2 and F3 mice generated from breeding F1 and F2 animals with naive controls had the same behavioral abnormalities (Franklin & Mansuy, 2011; Franklin et al., 2010; Weiss et al., 2011) which were observed for both male (Franklin et al., 2010) and female mice (Weiss et al., 2011). Mechanistically, these mice had an altered serotonergic signaling with a decrease in serotonin receptor 5HT1A binding in the dorsal raphe nuclei (Franklin & Mansuy, 2011). In addition, epigenetic processes were involved in the inheritance of these behavioral alterations since the F3 generation and the germline it originates from were never exposed to stress. In the F1 sperm cells, there were differences in DNA methylation patterns at the promoter regions of several genes implicated in the stress response, such as corticotrophin-releasing factor receptor 2 (CRFR2), cannabinoid receptor 1 (CB1), and methyl-CpG-binding protein 2 (MeCP2) (Franklin et al., 2010). Positive environmental experiences were also shown to be associated with epigenetic variations that result in long-term cognitive effects such as enhanced memory performance and synaptic plasticity. For instance, total levels of H3 and H4 acetylation and methylation were increased in the hippocampus and cortex of mice living in larger cages with a running wheel and in which toys were frequently rotated (Fischer, Sananbenesi, Wang, Dobbin, & Tsai, 2007). Moreover, these positive behavioral changes were also transmitted to subsequent generations through the mother and were independent of maternal care (Arai, Li, Hartley, & Feig, 2009). Most available studies, on transgenerational epigenetic inheritance, have focused on understanding the role of maternal transmission of environmentally induced epigenetic modifications; studies on the effects of paternal epigenetic alterations on offspring are still scarce. In a recent study by Petropoulos, Matthews, and Szyf (2014), it was reported that the administration of the synthetic glucocorticoid, dexamethasone, to adult male mice, leads to alterations of the DNA methylation patterns in the mature sperm cells of these

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animals which is associated with alterations in the expression and DNA methylation of nuclear steroid receptors (i.e., mineralocorticoid receptor, estrogen alpha receptor, and GR) in somatic tissues such as hippocampus and kidney of offspring (Petropoulos et al., 2014). It seems clear that several inherited brain disorders such as autism, fragile X, Angelman’s, Prader– Willi, Rett, and Beckwith–Wiedeman syndromes manifest during postnatal brain development and are thought to be due to epigenetic dysregulations (Falls, Pulford, Wylie, & Jirtle, 1999; Schanen, 2006). For instance, Rett syndrome, the most common form of autism in girls, arises from a mutation of MeCP2, a methyl-CpG-binding protein-2 gene, a key mediator of epigenetic repression of gene expression (Amir et al., 1999). Fragile X syndrome, a common form of mental retardation, arises from the hypermethylation of CGG triplet repeats located in the 50 -untranslated region of the fragile X mental retardation-1 (FMR1) gene and the loss of FMR1 protein expression ( Jin & Warren, 2000). Although in the case of the other inherited syndromes such as Angelman’s, Prader–Willi, and Beckwith–Wiedemann no direct association can be made between specific genomic lesions and susceptibility to the disorder, selected studies show that deletions of specific imprinted genes are involved and parent-of-origin affects are observed (Falls et al., 1999; Schanen, 2006). In addition, drug addiction and alcoholism in particular are the most serious substance abuse disorders worldwide. Alcohol drinking has been recognized as having several adverse health consequences, such as an increased risk of cardiovascular and liver diseases, cancer, neurobiological disorders, and fetal abnormalities (Edwards et al., 1996). Human genome-wide association studies of individuals with family history of alcoholism and twin studies have shown that several susceptibility genes are linked to the vulnerability and risk of developing alcohol-related disorders. However, alcoholism is a multifactorial disorder in which complex gene-to-gene and gene-to-environment interactions occur resulting in a variety of addiction phenotypes, such as “alcohol dependence” and “alcoholism or depression” phenotype (Corbin, Farmer, & Nolen-Hoekesma, 2013), and in neurological deficiencies. Environmental factors play an equally important role in the development of alcohol-related disorders, i.e., stressful life events have been shown to influence alcohol drinking and relapse behaviors (Corbin et al., 2013). Epigenetic mechanisms are believed to be involved in the alcohol’s deleterious effects on brain function and behavior and are thought to be responsible for the long-term effects of alcohol and for the transgenerational inheritance of these behaviors by offspring (Vassoler & Sadri-Vakili, 2013).


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Pregnancy is a particularly sensitive period to alcohol’s teratogenic effects on the developing fetal brain (Guerri, Bazinet, & Riley, 2009; Shukla et al., 2008). Alcohol consumption during pregnancy can result in a continuum of adverse outcomes to the fetus known as FASD. These disorders range from growth retardation to neurobehavioral alterations (i.e., poor stress tolerance), immune and endocrine system abnormalities, and higher incidence of cancers (Gauthier, Drews-Botsch, Falek, Coles, & Brown, 2005; Kelly et al., 2009; Latino-Martel et al., 2010; Schneider, Moore, Kraemer, Roberts, & DeJesus, 2002). Animal models of fetal alcohol exposure also show similar disorders such as, anxiety, stress hyperresponsiveness, immune deficiencies, and cancer susceptibility (Boyadjieva et al., 2009; Hellemans, Sliwowska, Verma, & Weinberg, 2010; Murugan, Zhang, Mojtahedzadeh, & Sarkar, 2013; Polanco, Crismale-Gann, Reuhl, Sarkar, & Cohick, 2010; Sarkar & Boyadjieva, 2007; Ting & Lautt, 2006). Our group have shown, using β-endorphin cell replacement studies, that a Pomc gene expression deficiency was associated with some of the pathologies observed in the FASD (Boyadjieva et al., 2009; Sarkar et al., 2007). In the hypothalamus, Pomc gene produces melanocortin and β-endorphin, two neuropeptide hormones that play an important role in energy homeostasis, stress response, immune fuction, and brain reward system (Cone, 2005; Luger, Scholzen, Brzoska, & Bohm, 2003; Mountjoy, 2010; Sarkar, Murugan, Zhang, & Boyadjieva, 2012; Smart, Tolle, Otero-Corchon, & Low, 2007). Epigenetic alterations are believed to be involved in the detrimental effects of alcohol on the fetal brain. Garro and colleagues (Garro, McBeth, Lima, & Lieber, 1991) were the first to show that the administration of ethanol to pregnant female mice on gestation days 9–11 results in a global hypomethylation of fetal DNA which was associated with a reduced level of DNA methyltransferases. Recently, we reported that fetal exposure to ethanol induced a longlasting hypermethylation of the proximal region of Pomc gene promoter which was associated with reduced Pomc gene expression and β-endorphin protein production (Govorko et al., 2012). These changes were associated with decreased protein and mRNA levels of histone activation marks (H3K4me3, Set7/9, acetylated H3K9, and phosphorylated H3S10), while it increased the repressive marks (H3K9me2, G9a, and Setdb1), DNAmethylating enzyme (Dnmt1), and the methyl-CpG-binding protein (MeCP2) in the hypothalamus of both male and female offspring of F1 generation (Bekdash et al., 2013; Govorko et al., 2012). Moreover, suppression of histone deacetylation and DNA methylation by pharmacological agents normalized Pomc gene expression and POMC neuronal function. We also

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tested whether these ethanol-induced Pomc gene epigenetic changes were transgenerationally transmitted across generations. We compared two different germlines, a male germline, which was produced by breeding male F1 fetal ethanol-exposed rats and their male offspring with naı¨ve females, and a female germline by breeding female fetal alcohol-exposed rats and their female offspring with naı¨ve males. The results are summarized in Fig. 2.2 and briefly described below. We found that the Pomc gene epigenetic changes were transgenerationally transmitted through the male germline to subsequent generations. F1, F2, and F3 male offspring had a significantly increased DNA methylation pattern; however, the female progeny showed these changes in Pomc gene methylation and expression only in the F1 generation but not in the F2 and F3 (Govorko et al., 2012). In addition, this Pomc gene methylation status was observed in sperm cells of the F1, F2, and F3 Alcohol-exposed mother

Hypothalamus F0

Fetal alcohol exposed F1 fetus and germline

Pomc gene repression

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SAM F1

HDACs

Dnmts Hypermethylation

Transgenerational epigenetic inheritance through male germline

Stress intolerance, immune deficiency, cancer susceptibility

b-endorphin neurons apoptosis

Ac

Epigenetic modifications

Deacetylation

CH3 CH3 CH3 CH3 CH3

F1 CpG island

Pomc gene promoter F2

F3

Figure 2.2 Transgenerational inheritance of the alcohol-induced epigenetic modifications of Pomc gene. Alcohol feeding to a pregnant rat leads to epigenetic changes (i.e., hypermethylation and deacetylation) in the Pomc gene promoter, Pomc gene silencing, and β-endorphin protein deficiency in the hypothalamus of F1 males and females. These effects were associated with stress intolerance, immune deficiencies, and cancer susceptibility. These epigenetic modifications were transgenerationally transmitted to F2 and F3 offspring through male germline.


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generations of male rats. Alcohol consumption was also shown to decrease the availability of the methyl-donor, S-adenosylmethionine (SAM). SAM is critical for DNA and histone methylation, indispensable to normal embryonic development and normal brain development (Kim et al., 2009; Robertson & Wolffe, 2000). SAM formation is dependent on the availability of major nutrients such as choline. Choline deficiency causes neurodevelopmental abnormalities such as neural tube defects in mice and in humans (Fisher, Zeisel, Mar, & Sadler, 2002; Shaw, Carmichael, Yang, Selvin, & Schaffer, 2004; Zeisel, 2006) and has negative effects on neuronal migration, survival, and differentiation (Craciunescu, Albright, Mar, Song, & Zeisel, 2003; Zeisel, 2011). We were the first to report that gestational choline supplementation prevented the ethanol-induced epigenetic alterations of histone marks and Pomc gene methylation in β-endorphinproducing POMC neurons in the hypothalamus of adult offspring rats and normalized their stress response to an immune challenge (Bekdash et al., 2013). Several studies have also reported that paternal exposure to alcohol has deleterious effects on offspring and can cause defects ranging from low birth weight and retarded growth to congenital malformations and increased neonatal mortality across several generations (Friedler, 1996; Jamerson, Wulser, & Kimler, 2004). The timing of paternal exposure similarly impacts the severity of alcohol’s effects on the brain’s neurobehavioral and cognitive functions in offspring (Bielawski & Abel, 1997; Jamerson et al., 2004). In humans, it was reported that paternal alcohol consumption, especially in the case of an early-onset alcoholism, has detrimental effects on behavioral and cognitive performances in the offspring (Poon, Ellis, Fitzgerald, & Zucker, 2000; Tarter, Jacob, & Bremer, 1989), and in addition to deficits in cognition and attention, children of alcoholic fathers have increased hyperactivity (Goodwin, Schulsinger, Hermansen, Guze, & Winokur, 1975). The role of sperm epigenetic modifications in these transgenerational effects of alcohol exposure was also suggested in numerous studies. For instance, it was shown that fetal exposure to alcohol reduces DNA methylation of the paternally imprinted growth-related gene H19 in the sperm of exposed mice (F1; Stouder, Somm, & Paoloni-Giacobino, 2011) and that in the same CpG sites in the brain, a reduced DNA methylation pattern was observed in F2 offspring. In the F2 male mice, a 26% decrease in sperm concentration was observed. These researchers argued that since H19 CTCFbinding sites are important for the regulation of Igf2 gene expression, the hypomethylation of H19 may be involved in the decreased spermatogenesis

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in offspring (Stouder et al., 2011). Findings from a human study, similarly found that moderate and heavy drinkers, compared with nondrinkers, had subtle reductions in DNA methylation at the H19 imprinted gene in sperm cells (Ouko et al., 2009). In another study on male rats, it was shown that paternal exposure to ethanol significantly decreased cytosine methyltransferase (Dnmt1) mRNA levels in sperm cells, which was suggested to be one of the mechanisms by which alcohol exposure induces changes in gene expression levels after conception (Bielawski, Zaher, Svinarich, & Abel, 2002). These studies clearly suggest that paternal exposure to alcohol can be detrimental to the offspring. sncRNA-dependent mechanisms were also involved in the ethanol’s teratogenic effects on brain cells (Balaraman, Tingling, Tsai, & Miranda, 2013; Wang et al., 2009). The initial studies investigating the interactions between ethanol and miRNA in fetal neuronal stem cells showed that high concentrations of ethanol significantly suppressed the expression of four miRNAs, miR-21, miR-335, miR-9, and miR-153, whereas lower concentration induced miR-335 expression (Sathyan, Golden, & Miranda, 2007). Moreover, miRNAs were also suggested to play an important role in the etiology of alcoholism and alcohol withdrawal (Pietrzykowski et al., 2008).

7. CONCLUDING REMARKS The studies summarized herein provide strong evidence suggesting that early-life experiences and environmental exposure to toxins, stress, or drugs of abuse, such as alcohol, can produce alterations in epigenetic programming and affect the expression of developmentally important genes, eventually leading to physiological and behavioral defects. The most intriguing finding from these studies is the permanent transgenerational transmission of epigenetic marks across several generations. Although the molecular processes involved in this epigenetic inheritance are still not fully identified, understanding these transgenerational effects and determining the germline molecular mechanisms involved hold a great promise for the determination of disease susceptibility and eventually the prevention and cure of developmentally induced adult-onset diseases and brain disorders.

ACKNOWLEDGMENT This study was supported by NIAAA Grants 5R37 AA08757, AA11591, and U24 AA014811.


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