ALCOHOL RESEARCH: Current Reviews

In Utero Alcohol Exposure, Epigenetic Changes, and Their Consequences

Michelle Ungerer; Jaysen Knezovich, M.Sc.; and Michele Ramsay, Ph.D.

Exposure to alcohol has serious consequences for the developing fetus, leading to a Michelle Ungerer is a Masters range of conditions collectively known as fetal alcohol spectrum disorders (FASD). student and Jaysen Knezovich, Most importantly, alcohol exposure affects the development of the brain during critical M.Sc., is a Ph.D. student and periods of differentiation and growth, leading to cognitive and behavioral deficits. The molecular mechanisms and processes underlying the teratogenic effects of alcohol lecturer in the Division of Human exposure remain poorly understood and are complex, because the specific effects Genetics, School of Pathology, depend on the timing, amount, and duration of exposure as well as genetic Faculty of Health Sciences, susceptibility. Accumulating evidence from studies on DNA methylation and histone University of the Witwatersrand modification that affect chromatin structure, as well as on the role of microRNAs in and the National Health Laboratory regulating mRNA levels supports the contribution of epigenetic mechanisms to the Service in Johannesburg, development of FASD. These epigenetic effects are difficult to study, however, because South Africa. they often are cell-type specific and transient in nature. Rodent models play an important role in FASD research. Although recent studies using these models have Michele Ramsay, Ph.D., is a yielded some insight into epigenetic mechanisms affecting brain development, they professor in the Division of have generated more questions than they have provided definitive answers. Human Genetics, National Health Researchers are just beginning to explore the intertwined roles of different epigenetic Laboratory Service, School of mechanisms in neurogenesis and how this process is affected by exposure to alcohol, Pathology, Faculty of Health causing FASD. KEY WORDS: Prenatal alcohol exposure; fetal alcohol spectrum Sciences, and Interim Director disorders; epigenetics; epigenetic mechanisms; epigenetic modifications; brain development; cognitive deficits; behavioral deficits; DNA methylation; histone of the Syndey Brenner Institute modification; chromatin; microRNAs; rodent models for Molecular Bioscience at the University of the Witwatersrand, Johannesburg, South Africa.

lcohol exposure of the developing tant role in these processes. This article FASD embryo and fetus in utero can reviews the current knowledge regard- Ahave a wide range of detrimental ing the contributions of epigenetic FASD can be associated with a variety effects collectively referred to as fetal modifications to the manifestations of symptoms that differ widely in alcohol spectrum disorders (FASD). of FASD, much of which has been severity depending on the specific Researchers are intensively investigating obtained using rodent models in which conditions of alcohol exposure. The the mechanisms that may contribute to the timing, frequency, duration, and most severe outcome is fetal alcohol alcohol’s effects on the developing amount of alcohol exposure can be syndrome (FAS), which can manifest organism and to the resulting conse- tightly controlled. This discussion also variably with diverse combinations of quences, particularly with respect to touches on the concepts of develop- craniofacial, growth, central nervous the cognitive and behavioral deficits mental reprogramming, the role of system (CNS), and neurobehavioral associated with FASD. These studies preconception alcohol exposure, and abnormalities (Jones et al. 1973; Sampson have yielded increasing evidence that transgenerational transmission of the et al. 1997). Associated psychosocial epigenetic mechanisms play an impor- effects of alcohol exposure. problems include learning difficulties,

In Utero Alcohol Exposure, Epigenetic Changes, and Their Consequences 37 attention deficit–hyperactivity disorder the ADH1B locus that result in an maintained through two mechanisms; (ADHD), and mental retardation altered amino acid sequence and function structural chromatin modifications (Burd et al. 2003; O’Leary 2004). of the encoded enzyme can influence (i.e., DNA methylation and histone Given that alcohol consumption is the severity of the adverse effects on modifications) and RNA interactions voluntary, FASD is said to be the the developing fetus (i.e., teratogenesis) (i.e., the actions of non-coding RNAs most preventable cause of birth defects in different ethnic populations (for a [ncRNAs]). In eukaryotes, the genome and mental retardation. FASD is a review see Ramsay 2010). However, is present in the cell nucleus in the global health concern, and worldwide to date few studies have supported a form of chromatin—a DNA–protein approximately 1 to 3 per 1,000 births role for genetics in the development complex that packages DNA into a is thought to be suffering from FAS. of FASD. highly condensed form. The structural In the United States, FAS prevalence Rodent models have provided a building blocks of chromatin are the ranges between 0.5 and 2.0 per 1,000 valuable tool for investigating genetic nucleosomes, each of which consists live births (Abel 1995; May and Gossage influences on the observable outcomes of 147 base pairs of DNA wrapped 2001). The highest rates of FAS have (i.e., phenotypes) associated with FASD. around a core of 8 histone proteins been reported in mixed-ancestry com- For example, the effects of in utero (Ooi and Henikoff 2007). The octamer munities in the Western Cape of South alcohol exposure differ between inbred core comprises two copies each of his- Africa, where between 68.0 and 89.2 and selectively bred mice. These find- tone proteins H2A, H2B, H3, and per 1,000 school-age children display ings highlight the contribution of a H4. Moreover, the nucleosomes are FAS symptoms (May et al. 2007). genetic predisposition to the suscepti- connected with each other by a linker Between 5 and 10 percent of bility to the detrimental effects of histone H1 that offers stability to the offspring who have been exposed to prenatal ethanol exposure and provide packaged structure. Modifications of alcohol prenatally display alcohol- additional support for the importance the chromatin structure affect the first related developmental anomalies (Abel of genetic factors in the development step of gene expression (i.e., transcrip- 1995), with the severity of the out- of FASD (Boehm et al. 1997; Gilliam tion). ncRNAs, on the other hand, act come determined by the dose, timing, et al. 1989; Ogawa et al. 2005). at the posttranscriptional level. and duration of exposure (Padmanabhan Although studies have investigated and Hameed 1988; Pierce and West the genetic susceptibility to FASD, the Chromatin Remodeling 1986; Sulik 1984). However, the pro- underlying cause(s) of these disorders portion of affected offspring may be still remains unclear. The wide range considerably higher in unfavorable of clinical features observed in people dnA Modifications. Both DNA and circumstances, including instances of affected by in utero alcohol exposure protein components of the nucleosome malnutrition of the mother and thus, underlines the importance of investigat- are subject to a variety of modifications the fetus. The genetic makeup of both ing the mechanisms of alcohol-related that can influence chromatin mother and fetus, in conjunction with teratogenesis at a molecular level. Because conformation and accessibility. The best-characterized epigenetic mark, other factors (e.g., gender, diet, and FASD is a developmental abnormality, DNA methylation, involves the social environment), also plays an impor- disruptions in normal cellular differen- covalent addition of a methyl (CH ) tant role in the manifestation of FASD tiation driven by changes in gene 3 group to one of the four DNA (Chernoff 1980; Ogawa et al. 2005). expression that in turn are regulated The effects of prenatal alcohol expo- nucleotides (i.e., cytosine [C]) to by epigenetic mechanisms are most form 5-methylcytosine (5mC). In sure are more similar in identical (i.e., likely involved in FASD pathogenesis. monozygotic) twins than in fraternal eukaryotes, methylation usually affects (i.e., dizygotic) twins, suggesting a her- C that are followed by the nucleotide itable component (Abel 1988; Chasnoff Epigenetic Modifications guanine (G) (i.e., that are part of a 1985; Streissguth and Dehaene 1993). CpG dinucleotide) (Rodenhiser and Genetic studies have shown that differ- The term epigenetics, first defined by Mann, 2006). At these sites, enzymes ent variants of the genes encoding Waddington in 1942 (as reprinted in called DNA methyltransferases various alcohol-metabolizing enzymes— Waddington 2012), refers to the changes (DNMTs) mediate the methylation such as alcohol dehydrogenases (ADHs), in gene expression that occur without 1 genomic imprinting is a genetic phenomenon in which only a aldehyde dehydrogenases (ALDHs), changes in the DNA sequence itself. gene copy inherited from one parent is expressed in the offspring. and cytochrome P450 2E1 (CYP2E1)— Epigenetics plays a vital role in regulat- each person inherits two copies of each gene, one inherited from the mother and one inherited from the father. in general, both of in the mother and their offspring can ing key developmental events, allowing these gene copies can be active in the offspring. For some genes, affect alcohol metabolism and con- for tissue-specific gene expression, however, one gene copy is “shut off” by genomic imprinting, so tribute to subsequent alcohol-related genomic imprinting,1 and stem-cell that only the non-imprinted copy remains active. For certain genes, the nonimprinted, active copy is always the one inherited damage (Gemma et al. 2007; Warren maintenance. Tissue-specific gene from the mother, whereas for other genes the nonimprinted, and Li 2005). For example, variants at expression patterns are established and active copy is always the one inherited from the father.

38 Alcohol Research: Current Reviews of C residues, thereby acting as critical they can be removed again by specific modifications allow the zygote to give modulators of fetal development (Li enzymes. rise to the cellular lineages that will et al. 1992). For these DNA methy- These histone modifications, form the embryo. Reprogramming lation reactions, DNMTs use methyl together with DNA methylation, influ- occurs in two phases during in utero groups produced by a sequence of ence chromatin structure and have a development, one shortly after fertil- reactions known as the folate pathway profound influence on gene regulation. ization and the other in the developing (Friso et al. 2002). Generally, DNA Both of these types of epigenetic modi- gametes of the fetus. The first phase methylation is associated with a fications work together to remodel the takes place after fertilization in the condensed chromatin conformation, chromatin and partition the genome preimplantation embryo (i.e., the blas- which effectively silences gene into two different functional domains— tocyst). During this phase, embryonic expression because the enzymes transcriptionally active regions collectively epigenetic patterns are re-established in needed for transcription cannot known as euchromatin and transcrip- a lineage-specific manner in the inner access the DNA. tionally inactive regions collectively cell mass of the blastocyst (figure 1). More recent studies have found called heterochromatin. Euchromatic The second phase occurs in the that 5mC can be further modified by regions are modified to allow an open gametes, where rapid genome-wide enzymes called ten-eleven translocation conformation, rendering the regions demethylation is initiated to erase (Tet) proteins, in a process referred accessible to cellular proteins favoring existing parental methylation patterns, to as iterative oxidation. This results transcription. In contrast, heterochro- followed by re-establishment of epige- in the formation of several reaction matic regions, such as the ends of netic marks in a sex-specific manner products (i.e., derivatives), including chromosomes (i.e., telomeres) and (Reik et al. 2001). 5-hydroxymethylcytosine (5hmC), regions around the center of the chro- Researchers recently have begun 5-formylcytosine (5fC), and 5-car- mosome (i.e., pericentric regions), to investigate epigenetic mechanisms boxylcytosine (5caC) (Ito et al. 2011; generally exhibit a closed conformation as key contributors to the development Tahiliani et al. 2009). Although the that limits interactions between the of FASD. This research was prompted role of these methylation derivatives DNA and cellular proteins, thereby by the observation that periods of still remains unclear (Branco et al. 2012) silencing gene activity (for a review, see increased vulnerability to in utero they seem to serve different functions Schneider and Grosschedl 2007). alcohol exposure coincide with those than 5mC. Thus, the conversion of Additionally, chromatin structure, and of reprogramming events. In addition, 5mC to 5hmC has been implicated in thus gene expression, is influenced by evidence suggests that environmental active DNA demethylation (Wu and the specific combination of histone factors, and specifically alcohol, are Zhang, 2010). Furthermore, whereas variants in a nucleosome, the spacing able to alter epigenetic modifications. 5mC typically is found in regions reg- between nucleosomes (i.e., nucleosome This provides a link between the geno- ulating the expression of specific genes occupancy), and the position of each type, environment, and disease. (i.e., in promoters), 5hmC is associated nucleosome within the nucleus (i.e., with the bodies of the affected genes or nuclear architecture) (Cairns 2009). with promoters of developmental regu- Alcohol and Biological latory genes (Wu et al. 2011). Finally, Pathways 5hmC appears to play an important Developmental Reprogramming role in reprogramming the paternal As mentioned previously, DNA genome following fertilisation (Hackett Epigenetic reprogramming is a process methylation reactions rely on the and Surani 2013). (Reprogramming that involves the erasure and then folate pathway to supply the necessary will be discussed in the following section.) re-establishment of chromatin modifica- methyl groups. Excessive alcohol expo- tions during mammalian development. sure is known to interfere with normal Histone Modifications. The histones It serves to erase random changes in folate metabolism and reduce its making up the core of the nucleosome epigenetic marks (i.e., epimutations) bioavailability (Halsted and Medici have unstructured N-terminal tails that have occurred in the germ cells 2012). Folate is required as a coenzyme that protrude from the nucleosome and (i.e., gametes) and to restore the ability to supply methyl groups needed for which are subject to modifications. of the fertilized egg cell (i.e., zygote) to the formation of a compound called Histone modifications are varied and develop into all the different cell types S-adenosylmethionine (SAMe), which include acetylation, methylation, and tissues (Reik et al. 2001). Epigenetic in turn participates in reactions in phosphorylation, ubiquitinylation, modifications are modulated in a tem- which the methyl group is transferred ADP-ribosylation, and sumoylation poral and spatial manner and act as to another molecule (i.e., transmethy- at specified residues (for a review, see reversible switches of gene expression lation reactions). In the folate-dependent Kouzarides 2007). Importantly, these that can lock genes into active or pathway, the enzyme methionine modifications are dynamic—that is, repressed states. In addition, these synthase (MS), which requires vitamin

in Utero Alcohol Exposure, Epigenetic Changes, and Their Consequences 39 B12 to function properly, is responsible al. 1993). Additionally, ethanol appears sure to alcohol in these animals results for transferring the methyl group con- to enhance the loss of methyl groups, in a wide range of anomalies, including tained within the 5-methyl-tetrahyrofolate which in turn disrupts subsequent growth retardation, CNS malformations, compound to homocysteine, which SAMe-dependent transmethylation mental disability, and distinct craniofa- ultimately generates methionine (Friso reactions (Schalinske and Nieman 2005). cial dysmorphology (Anthony et al. et al. 2002). The methionine is converted 2010; Boehm et al. 1997; Boggan et to SAMe by methionine adenosyltrans- al. 1979; Bond and Di Giusto 1977; ferase (MAT), and the SAMe then is Rodent Models of Prenatal Klein de Licona et al. 2009; McGivern used for the methylation of DNA. As Ethanol Exposure 1989; Parnell et al. 2009). early as 1974, research on alcohol-fed The FASD-like phenotypes observed rats described reduced MS activity and The teratogenic effects of prenatal in these rodent models have been asso- subsequent reduction of the levels of alcohol exposure have been examined ciated with alterations in global gene both methionine and SAMe (Barak et in rodent models for several decades. expression, particularly in the developing al. 1987; Finkelstein 1974; Trimble et Studies have shown that in utero expo- brain (Hard et al. 2005; Hashimoto-

Figure 1 Reprogramming in mammalian development. Two waves of epigenetic reprogramming occur during embryo development. The first phase of reprogramming occurs in the normal body cells (i.e., somatic cells) of the developing embryo. In mice, following fertilization, the embryo undergoes genome-wide demethylation that is completed by embryonic day 5 (E5). The paternal genome (blue line) undergoes rapid, active demethylation, whereas in the maternal genome (pink line), demethylation occurs via a passive process. Remethylation of the embryonic genome begins at day E5 and is completed prior to birth. The second wave of epigenetic reprogramming occurs in the germ cells of the developing embryo, which will ultimately give rise to gametes that contain sex-specific epigenetic signatures. The primordial germ cells (PGCs) of the developing embryo contain the methylation signatures of the parental genomes. At approximately E7–8, the PGCs undergo rapid demethylation that is complete by E15–16. Following this, sex-specific methylation is re-established. In the male germline, reprogramming is complete at birth (blue line), whereas in females, reprogramming continues until puberty (pink line).

SOURCE: Adapted from Reik et al. 2001; Smallwood and Kelsey 2012.

40 Alcohol Research: Current Reviews Torii et al. 2011, 2011; Kleiber et al. Avy is a dominant mutation that is with changes in DNA methylation and 2012). This association, in conjunc- caused by the insertion of a DNA concurrent alterations in the expression tion with the vital role that epigenetic sequence known as an intracisternal of other genes. Downing and colleagues mechanisms play in controlling gene A-particle (IAP) retrotransposon in (2010) found that in utero ethanol expression, suggests that normal epige- front of (i.e., upstream of) the Agouti exposure resulted in reduced methylation netic regulation by DNA methylation, gene. Animals that carry one mutant in the embryo at the Igf2 locus, which histone modifications, and ncRNAs is and one wild-type gene copy (i.e., het- encodes insulin-like growth factor 2, disrupted as a result of ethanol insult. erozygous Avy/a mice) display a variety with a concomitant change in Igf2 of coat colors, ranging from yellow to gene expression. These changes in gene Prenatal ethanol exposure and mottled and brown, even though they expression were accompanied by skeletal dnA Methylation are genetically identical. Avy expression malformations similar to those observed is strongly correlated to the DNA in FAS patients. In other studies, alcohol A direct link exists between ethanol methylation profile of the inserted exposure resulted in neural tube defects exposure and aberrations in DNA IAP. If the IAP shows hypomethylation, in conjunction with genome-wide methylation. For example, in a mouse the Agouti gene is constantly expressed bidirectional methylation changes (i.e., model evaluating the effects of in utero (i.e., shows constitutive ectopic Agouti occurrence of both hypo- and hyper- ethanol exposure from days 9 to 11 of expression) and the animals have a yellow methylation) (Liu et al. 2009). These gestation, this acute ethanol adminis- coat. Conversely, hypermethylation altered methylation profiles were asso- tration resulted in lower-than-normal correlates with promoter silencing and ciated with significant changes in the methylation throughout the genome a pseudoagouti coat (Dolinoy et al. expression of several genes associated (i.e., in global hypomethylation) of 2010). Kaminen-Ahola and colleagues with multiple functions, including fetal DNA (Garro 1991). Further- (2010) investigated the effect of gesta- chromatin remodeling, neuronal more, the ethanol-exposed fetuses vy tional ethanol exposure in A het- morphogenesis, synaptic plasticity, displayed significantly reduced levels erozygous mice, demonstrating that and neuronal development. of DNA methylase activity. Ethanol- ethanol exposure increased the propor- Together, these findings provide induced reductions in DNA methyla- tion of pseudoagouti-colored offspring. compelling evidence for alcohol- tion affect not only the fetus but also This change in the proportion of coat induced alterations of DNA methylation the placenta in pregnant mice exposed colors was linked to transcriptional patterns in exposed fetuses that elicit a to alcohol (Haycock and Ramsay 2009). silencing of the mutant allele, which in phenotype that is at least in part similar More recently, researchers evaluated turn correlated with hypermethylation to that observed in FASD. the effect of prenatal alcohol exposure of the Avy locus. This study highlights on DNA methylation of five imprinted the ability of prenatal alcohol exposure genes in male offspring; these analyses Prenatal ethanol exposure and to alter the fetal epigenotype (albeit Histone Modifications detected a decrease in DNA methylation only at a specific locus) and, conse- at a single locus in the H19 imprinting quently, the adult phenotype. Rodent models of alcoholism and in control region in the sperm of these In addition to the aberrant expres- utero exposure to ethanol, as well as males (Stouder et al. 2011). Finally, in sion at the Agouti locus in the Avy het- studies using cultured cells (i.e., in utero ethanol exposure in mice hinders erozygous mice, Kaminen-Ahola and vitro experiments) have provided sig- the acquisition of DNA methylation in colleagues (2010) noted altered gene nificant insights into the effects of a brain region called the dentate gyrus, expression profiles in the livers of their alcohol on protein modifications, par- which is associated with developmental ethanol-exposed wild-type (a/a) siblings, ticularly to histones. Excess alcohol retardation (Chen et al. 2013). as well as growth restriction and certain intake can exert its effect on protein Other analyses have looked at craniofacial dysmorphologies that are modifications either directly or indirectly methylation patterns of specific genes reminiscent of human FAS symptoms. by disrupting the epigenetic machinery. rather than global DNA methylation. Together, the findings that ethanol As with DNA methylation, some For example, a gene called Agouti has exposure can alter DNA methylation of these mechanisms involve folate, which been used extensively as a model to at the Agouti locus and elicit an associated as mentioned earlier serves as methyl study the effects of environmental (i.e., phenotype (i.e., altered coat color), group donor for histone methylation. dietary) exposures on DNA methylation. suggests that other epigenetic targets Folate deficiency is a common clinical The murine Agouti (A) locus regulates and associated gene expression also sign of chronic alcohol abuse and has the animals’ coat color; animals that may be disrupted and may be responsi- been implicated in the development of carry two copies of the common vari- ble for the occurrence of a phenotype alcoholism-related complications, such ant, referred to as the wild-type allele, that corresponds to FAS in humans. as alcoholic liver disease (Eichner et al. (i.e., a/a mice) display a pseudoagouti Similar studies have demonstrated 1971). These deficiencies have been (i.e., brown) coat. A gene variant called the association of ethanol exposure associated with significant alterations

in Utero Alcohol Exposure, Epigenetic Changes, and Their Consequences 41 in histone modifications, particularly both H3 acetylation and significant in insulin signaling. People with FAS at lysine residues (Esteller 2008; Kim gene-expression changes. These findings often exhibit an underdeveloped cere- and Shukla 2005; Park et al. 2003; Shukla suggest that alterations to histone mod- bellum (i.e., cerebellar hypoplasia) et al. 2008). Altered histone modifica- ifications are a potential mechanism for that is associated with impaired insulin- tion, in turn, is associated with altered alcohol-induced cardiac disease (Zhong stimulated survival signaling. This gene expression (Pal-Bhadra et al. 2007). et al. 2010). An additional study by impaired signaling is mediated by the In in vitro studies using cultured rat Guo and colleagues (2011) assessed the body’s inability to properly respond to liver cells (i.e., hepatocytes), ethanol effects of alcohol on histone modifications insulin (i.e., insulin resistance) (Soscia exposure has been associated with bidi- in the cerebellum. The investigators et al. 2006). It has been posited that rectional changes in histone methylation, chronic in utero ethanol exposure including increased methylation at produces both insulin resistance in lysine 4 of histone H3 (i.e., increased the CNS and oxidative stress, which H3K4me2) and decreased methylation is thought to play a major role in at lysine 9 of histone H3 (i.e., decreased Another epigenetic alcohol-related neurobehavioral terato- H3K9me2) (Pal-Bhadra et al. 2007). mechanism by which genesis (de la Monte and Wands 2010). In addition, ethanol exposure led to alcohol could exert an In an in vivo model, adult rats prenatally selective acetylation of H3K9 (Park effect on the epigenome exposed to alcohol exhibited reduced et al. 2003). These findings have been insulin signaling and increased expres- supported by in vivo models that have is though the action of sion of genes that regulate insulin (i.e., demonstrated increased H3K9 acetyla- microRNAs (miRNAs). genes encoding proteins called TRB3 tion in the liver, lung, and spleen of and PTEN) in the liver (Yao and adult rats acutely exposed to alcohol Nyomba 2008). The analyses further (Kim and Shukla 2006). Chronic alcohol suggested that the observed hepatic exposure in adult rats also has been insulin resistance induced by alcohol associated with increases in histone found that perinatal alcohol exposure exposure was associated with reduced H3 and H4 acetylation in the amyg- decreased the expression and function acetylation of the TRB3 and PTEN dala of the brain that subsequently led of one type of histone acetyl transferase proteins. to changes in the expression of the called CREB binding protein (CBP). Taken together these findings gene encoding a signaling molecule Altered CBP function resulted in decreased suggest that alcohol-induced protein, known as neuropeptide Y (Pandey et lysine acetylation on histones H3 and and particularly histone, alterations al. 2008). This increase in acetylation H4 within the cerebellum, which may continue to provide alternative or may result either from a decrease in the contribute to the motor-activity deficits additional layers of complexity to activity of the enzyme that removes observed in FAS/FASD patients. an epigenetic etiology for FASD. acetyl groups (i.e., histone deacetylase) More recently, researchers investi- or an increase in the activity of the gated the effects of alcohol exposure on Prenatal ethanol exposure and enzyme that adds acetyl groups (i.e., certain fetal neuronal stem cells (i.e., ncRnA dysregulation histone acetylase). Finally, in utero mod- fetal cerebral cortical neuroepithelial els have revealed that embryos exposed stem cells) and associated gene expression. Another epigenetic mechanism by to acute levels of alcohol at mid-gestation These analyses found that ethanol which alcohol could exert an effect on showed elevated H3K9/18 acetylation exposure led to significant reductions the epigenome is though the action of as well as increased programmed cell in the levels of H3K4me3 (which acti- microRNAs (miRNAs). These small death, referred to as apoptosis, of the vates gene expression) and H3K27me3 ncRNAs play a critical role in several fetal lung (Wang et al. 2010). (which represses gene expression) (Veazey key biological processes, especially dur- Zhong and colleagues (2010) inves- et al. 2013). Despite the reduction in ing in utero development, including tigated the effects of high and low lev- expression-activating H3K4me3 levels, cell-cycle regulation, differentiation, els of alcohol exposure on H3 acetyla- both increased and decreased transcription and organ formation (i.e., organogene- tion and subsequent expression of was observed in the genes investigated. sis). Individual miRNAs can affect genes related to heart development Furthermore, loss of the repressive many target genes, silencing their (i.e., GATA4, Mef2c, and Tbx5) in methylation mark, H3K27me3, did expression either by preventing transla- cardiac progenitor cells. Results indicated not result in altered transcription levels. tion of the intermediate molecules that low levels of alcohol increased H3 Altered protein modifications in (i.e., messenger RNAs [mRNAs]) that acetylation but did not significantly response to alcohol exposure also may are generated during transcription or change the expression of the heart- involve proteins other than histones by causing mRNA cleavage. Experimental development–related genes. In con- that contribute to other manifestations evidence indicates that the expression trast, high levels of alcohol induced of FAS, including proteins involved of miRNAs is altered following exposure

42 Alcohol Research: Current Reviews to alcohol during development, and sion of these two miRNAs can disrupt on offspring development (Knezovich this may be one of the mechanisms developmental processes because they and Ramsay 2012). underlying alcohol-related teratogenesis are thought to regulate expression of a (Sathyan et al. 2007; Wang et al. 2009). group of genes called the Hoxb gene miRNAs have been implicated in family (Wang et al. 2009). This group Transgenerational Transmission the development of brain damage in of genes is involved in the regulation of the Effects of Alcohol Exposure response to prenatal alcohol exposure. and establishment of body patterning Miranda and colleagues (2010) have during embryonic development. Altered epigenetic modifications (i.e., hypothesized that ethanol causes brain Interestingly, there was no overlap in epimutations) may also be passed on damage during development by pro- the miRNAs between this study and from one generation to the next. There moting the cell cycle of neural stem those identified in the study by Sathyan are two modes in which such a trans- cells. This would accelerate the matu- and colleagues (2007), suggesting that mission of epimutations can occur ration of these progenitor cells and different models for alcohol exposure (Skinner 2008): result in their premature depletion. as well as the investigation of different This hypothesis is compatible with tissues and different developmental • Multigenerational inheritance, in the observation that when clusters of time periods of exposure may have which several generations are neural stem cells (i.e., neurospheres) varying impacts on diverse miRNA affected because they all are exposed are grown in culture, differentiating targets. neuroblasts from these clusters show Taken together, the preliminary to the same factor (e.g., alcohol) increased migration and depletion of studies suggest that miRNA plays a and thus are prone to the same stem cells when they are exposed to crucial role in normal development modifications; and ethanol compared with their unexposed and that this process can be disrupted counterparts. This observed behavior by alcohol exposure during critical • Transgenerational inheritance, suggests the involvement of a large periods, especially during neurogenesis. which involves a reprogramming network of genes controlling complex event in the germline in response biological outcomes. In order to exam- to a specific factor (e.g., alcohol ine the trigger for this behavior, Role of Preconception Alcohol exposure), resulting in an altered researchers examined miRNA expression Exposure in FASD epigenome that would be inherited levels in alcohol-exposed and nonex- by future generations even if they posed neural stem cells. A preliminary Although studies of FASD etiology are not themselves exposed to the screen of miRNAs in neural stem cells predominantly have focused on maternal same factor. identified four miRNAs (i.e., miR9, exposure during pregnancy, evidence miR21, miR135 and miR355) that also exists in support of contributions It was previously believed that were suppressed in the presence of of paternal exposure. For example, transgenerational epigenetic inheritance ethanol exposure (Sathyan et al. 2007). FAS-like effects have been observed would be unlikely because, as mentioned These miRNAs were found to act both in children of alcoholic fathers even previously, epigenetic reprogramming antagonistically and synergistically, in the absence of gestational alcohol occurs in the germline. However, both reducing and promoting apoptosis. exposure, suggesting the possibility increasing evidence indicates that Normally, these miRNAs favor normal that preconception alcohol exposure transgenerational epigenetic inheritance development by balancing cell survival may affect offspring development does indeed happen (Anway et al. and cell proliferation. Following alcohol (Abel and Tan 1988; Lemoine et al. 2005; Crepin et al. 2012; Stouder and exposure, however, the reduction in 1968). Studies conducted in rodents Paoloni-Giacobino 2010). Most of the their levels leads to an imbalance with 100 years ago have supported these detrimental effects. findings (Stockard 1913; Stockard and work conducted thus far in this area In another study (Wang et al. 2009), Papanicolaou 1916), and more recent has focused on the effects of agents pregnant mice were exposed to ethanol analyses also reported that paternal that can interfere with the body’s normal from day 6 to day 15 of gestation, and preconception alcohol exposure was hormone systems (e.g., vinclozolin, fetal brain tissue was examined for dif- associated with neurobehavioral abnor- which affects sex hormone levels and ferential miRNA expression. Under malities, low birth weights, congenital has been shown to have transgenera- these conditions, seven miRNAs were malformations, and growth retardation tional effects). The potential transgen- upregulated and eight were downregu- in offspring (Friedler 1996; Jamerson erational effects of alcohol and their lated in response to ethanol exposure, et al. 2004). Additional studies have role in the etiology and perpetuation with miR10a and miR10b showing implicated a role for altered sperm DNA of FAS/FASD symptoms in affected the highest level of overexpression. It is methylation in paternally-mediated individuals and their progeny, however, biologically plausible that overexpres- effects of preconception ethanol exposure still need to be determined.

in Utero Alcohol Exposure, Epigenetic Changes, and Their Consequences 43 Conclusions plex picture of locus-specific and cell- effects of alcohol exposure on epige- type–restricted effects is emerging. In netic regulatory mechanism and their Evidence is rapidly accumulating in particular, many studies have focused association with FAS-like symptoms. support of an epigenetic etiology in the on ethanol’s effects on mechanisms Drinking patterns in pregnant women, development of FASD (figure 2). All that regulate neurogenesis, leading to in contrast, are seldom accurately doc- three types of epigenetic modulators— the most devastating consequences of umented and often occur throughout DNA methylation, histone modifications alcohol exposure during development. gestation, which, not surprisingly, leads and regulation by ncRNAs—are per- to a vast array of phenotypes now rec- turbed by ethanol exposure. These The range of effects appears to be sig- nificantly influenced by the timing and ognized under the banner of FASD. ethanol-related changes can affect gene Thus, discerning the role of epigenetic expression of critical developmental level of exposure, leading to a wide range of outcomes and combinations mechanisms in these processes will be genes and pathways, impacting cell much more challenging. ■ proliferation and differentiation. of phenotypic indicators. The phenotypic consequences of in In mouse models, ethanol exposure utero ethanol exposure are significantly can be carefully controlled and other Acknowledgments correlated with the molecular conse- environmental parameters, such as diet quences of ethanol’s effects on epige- and stress, can be kept constant. This Michelle Ungerer and Jaysen Knezovich netic regulatory mechanisms. A com- allows for careful investigation of the contributed equally to writing this review.

Figure 2 Epigenetic contributions to FASD. Following conception, a complex orchestration of epigenetic mechanisms ensures normal cellular differ- entiation and embryonic development (green horizontal arrow). These mechanisms include DNA methylation, histone modifications, and non-coding RNAs (ncRNAs) to modulate gene expression in a specified temporal and spatial manner. Alcohol exposure in utero (red downward arrow) has been shown to alter these epigenetic modulators, which may consequently dysregulate gene expression patterns as indicated by the study findings listed and affect normal embryonic development and phenotype outcome. By these mechanisms, alcohol- induced epigenetic aberrations may contribute to the etiology of fetal alcohol spectrum disorders (FASD).

44 Alcohol Research: Current Reviews Financial Disclosure ChernoFF, g.F. The fetal alcohol syndrome in mice: tions of the royal society of london. series B, Biological Maternal variables. Teratology 22(1):71–75, 1980. Sciences 368(1609):20110328, 2013. PMid: 23166392 PMid: 7003793 The authors declare that they have no halsTed, C.h., and MediCi, V. aberrant hepatic methionine competing financial interests. CrePin, M.; dieu, M.C.; leJeune, s.; eT al. evidence of con- metabolism and gene methylation in the pathogenesis stitutional Mlh1 epimutation associated to transgenera- and treatment of alcoholic steatohepatitis. International tional inheritance of cancer susceptibility. Human Journal of Hepatology 2012:959746, 2012. PMid: Mutation 33(1):180–188, 2012. PMid: 21953887 22007317 References de la MonTe, s.M., and Wands, J.r. role of central ner- hard, M.l.; aBdolell, M.; roBinson, B.h.; and koren, g. vous system insulin resistance in fetal alcohol spectrum gene-expression analysis after alcohol exposure in the aBel, e.l. Fetal alcohol syndrome in families. Neuro - disorders. Journal of Population Therapeutics and developing mouse. Journal of Laboratory and Clinical toxicology and Teratology 10(1):1–2, 1988. PMid: 3352564 Clinical Pharmacology 17(3):e390–404, 2010. PMid: Medicine 145(1):47–54, 2005. PMid: 15668661 aBel, e.l. an update on incidence of Fas: Fas is not an 21063035 hashiMoTo-Torii, k.; kaWasaWa, Y.i.; kuhn, a.; and rakiC, P. equal opportunity birth defect. Neurotoxicology and dolinoY, d.C.; Weinhouse, C.; Jones, T.r.; eT al. Variable Combined transcriptome analysis of fetal human and Teratology 17(4):437–443, 1995. PMid: 7565490 histone modifications at the avy metastable epiallele. mouse cerebral cortex exposed to alcohol. Proceedings aBel, e.l., and Tan, s.e. effects of paternal alcohol con- Epigenetics 5(7):637–644, 2010. PMid: 20671424 of the National Academy of Sciences of the United sumption on pregnancy outcome in rats. Neurotoxicology States of America 108(10):4212–4217, 2011. PMid: doWning, C.; Johnson, T.e.; larson, C.; eT al. subtle 21368140 and Teratology 10(3):187–192, 1988. PMid: 3211095 decreases in dna methylation and gene expression at anThonY, B.; VinCi-Booher, s.; WeTherill, l.; eT al. alcohol- the mouse igf2 locus following prenatal alcohol expo- haYCoCk, P.C., and raMsaY, M. exposure of mouse induced facial dysmorphology in C57Bl/6 mouse mod- sure: effects of a methyl-supplemented diet. Alcohol embryos to ethanol during preimplantation develop- els of fetal alcohol spectrum disorder. Alcohol 44(7–8): 45(1):65–71, 2011. PMid: 20705422 ment: effect on dna methylation in the h19 imprinting control region. Biology of Reproduction 81(4):618–627, 659–671, 2010. PMid: 20570474 eiChner, e.r.; PierCe, h.i.; and hillMan, r.s. Folate balance 2009. PMid: 19279321 anWaY, M.d.; CuPP, a.s.; uzuMCu, M.; and skinner, M.k. in dietary-induced megaloblastic anemia. New England epigentic transgenerational actions of endocrine disrup- Journal of Medicine 284(17):933–938, 1971. PMid: iTo, s.; shen, l.; dai, q.; eT al. Tet proteins can convert 5- tors and male fertility. Science 308(5727):1466–1469, 5551802 methylcytosine to 5-formylcytosine and 5-carboxylcyto- sine. Science 333(6047):1300–1303, 2011. PMid: 2005. PMid: 15933200 esTeller, M. epigenetics in cancer. New England Journal 21778364 of Medicine 358(11):1148–1159, 2008. PMid: Barak, a.J.; BeCkenhauer, h.C.; TuMa, d.J.; and Badakhsh, s. effects of prolonged ethanol feeding on methionine 18337604 JaMerson, P.a.; Wulser, M.J.; and kiMler, B.F. neurobehavioral effects in rat pups whose sires were metabolism in rat liver. Biochemistry and Cell Biology FinkelsTein, J.d. Methionine metabolism in mammals: exposed to alcohol. Brain research. Developmental 65(3):230–233, 1987. PMid: 3580171 The biochemical basis for homocystinuria. Metabolism: Brain Research 149(2):103–111, 2004. PMid: 15063090 Clinical and Experimental 23(4):387–398, 1974. PMid: BoehM, s.l., 2nd; lundahl, k.r.; CaldWell, J.; and gilliaM, 4593752 Jones, k.l.; sMiTh, d.W.; ulleland, C.n.; and sTreissguTh, P. d.M. ethanol teratogenesis in the C57Bl/6J, dBa/2J, Pattern of malformation in offspring of chronic alcoholic and a/J inbred mouse strains. Alcohol 14(4):389–395, Friedler, g. Paternal exposures: impact on reproductive mothers. Lancet 1(7815):1267–1271, 1973. PMid: 1997. PMid: 9209555 and developmental outcome. An overview. Pharmacology, 4126070 Biochemistry, and Behavior 55(4):691–700, 1996. Boggan, W.o.; randall, C.l.; and dodds, h.M. delayed PMid: 8981601 kaMinen-ahola, n.; ahola, a.; Maga, M.; eT al. Maternal sexual maturation in female C57Bl/6J mice prenatally ethanol consumption alters the epigenotype and the exposed to alcohol. Research Communications in Friso, s.; Choi, s.W.; girelli, d.; eT al. a common muta- phenotype of offspring in a mouse model. PLoS Chemical Pathology and Pharmacology 23(1):117–125, tion in the 5,10-methylenetetrahydrofolate reductase Genetics 6(1):e1000811, 2010. PMid: 20084100 1979. PMid: 441506 gene affects genomic dna methylation through an inter- action with folate status. Proceedings of the National kiM, J.s., and shukla, s.d. histone h3 modifications in rat Bond, n.W., and di giusTo, e.l. Prenatal alcohol con- Academy of Sciences of the United States of America hepatic stellate cells by ethanol. Alcohol and Alcoholism sumption and open-field behaviour in rats: effects of 99(8):5606–5611, 2002. PMid: 11929966 40(5):367–372, 2005. PMid: 15939707 age at time of testing. Psychopharmacology 52(3):311– 312, 1977. PMid: 406636 garro, a.J.; MCBeTh, d.l.; liMa, V.; and lieBer, C.s. ethanol kiM, J.s., and shukla, s.d. acute in vivo effect of ethanol consumption inhibits fetal dna methylation in mice: (binge drinking) on histone h3 modifications in rat tis- BranCo, M.r.; FiCz, g.; and reik, W. uncovering the role implications for the fetal alcohol syndrome. Alcoholism: sues. Alcohol and Alcoholism 41(2):126–132, 2006. of 5-hydroxymethylcytosine in the epigenome. Nature Clinical and Experimental Research 15(3):395–398, PMid: 16314425 Reviews. Genetics 13(1):7–13, 2011. PMid: 22083101 1991. PMid: 1877725 kleiBer, M.l.; lauFer, B.i.; WrighT, e.; eT al. long-term alter- Burd, l.; MarTsolF, J.T.; klug, M.g.; and kerBeshian, J. geMMa, s.; ViChi, s.; and TesTai, e. Metabolic and genetic ations to the brain transcriptome in a maternal voluntary diagnosis of Fas: a comparison of the Fetal alcohol factors contributing to alcohol induced effects and fetal consumption model of fetal alcohol spectrum disorders. syndrome diagnostic Checklist and the institute of Medicine alcohol syndrome. Neuroscience and Biobehavioral Brain Research 1458:18–33, 2012. PMid: 22560501 Criteria for Fetal alcohol syndrome. Neurotoxicology and Reviews 31(2):221–229, 2007. PMid: 16908065 Teratology 25(6):719–724, 2003. PMid: 14624971 klein de liCona, h.; karaCaY, B.; MahoneY, J.; eT al. a single gilliaM, d.M.; koTCh, l.e.; dudek, B.C.; and rileY, e.P. exposure to alcohol during brain development induces Cairns, B.r. The logic of chromatin architecture and ethanol teratogenesis in selectivity bred long-sleep and microencephaly and neuronal losses in genetically sus- remodelling at promoters. Nature 461(7261):193–198, short-sleep mice: a comparison to inbred C57Bl/6J ceptible mice, but not in wild type mice. NeuroToxicology 2009. PMid: 19741699 mice. Alcoholism: Clinical and Experimental Research 30(3):459–470, 2009. PMid: 19442832 13(5):667–672, 1989. PMid: 2688466 ChasnoFF, i.J. Fetal alcohol syndrome in twin pregnancy. knezoViCh, J.g., and raMsaY, M. The effect of preconcep- Acta Geneticae Medicae et Gemellologiae (roma) 34(3- guo, W.; CrosseY, e.l.; zhang, l.; eT al. alcohol exposure tion paternal alcohol exposure on epigenetic remodel- 4):229–232, 1985. PMid: 3832736 decreases CreB binding protein expression and histone ing of the h19 and rasgrf1 imprinting control regions in acetylation in the developing cerebellum. PLoS One mouse offspring. Frontiers in Genetics 3:10, 2012. Chen, Y.; ozTurk, n.C.; and zhou, F.C. dna methylation 6(5):e19351, 2011. PMid: 21655322 PMid: 22371710 program in developing hippocampus and its alteration by alcohol. PLoS One 8(3):e60503, 2013. PMid: haCkeTT, J.a., and surani, M.a. dna methylation dynamics kouzarides, T. Chromatin modifications and their func- 23544149 during the mammalian life cycle. Philosophical transac- tion. Cell 128(4):693–705, 2007. PMid: 17320507

in Utero Alcohol Exposure, Epigenetic Changes, and Their Consequences 45 leMoine, P.; harousseau, h.; BorTeYru, J.P.; and MenueT, PierCe, d.r., and WesT, J.r. Blood alcohol concentration: in sperm. Reproductive Toxicology 31(4):507–512, J.C. les enfants des parents alcoholiques: anomolies a critical factor for producing fetal alcohol effects. 2011. PMid: 21382472 observees a propos de 127 cas (The children of Alcohol 3(4):269–272, 1986. PMid: 3638973 sTreissguTh, a.P., and dehaene, P. Fetal alcohol syndrome alcoholic parents: anomalies observed in 127 cases). raMsaY, M. genetic and epigenetic insights into fetal in twins of alcoholic mothers: Concordance of diagnosis Quest Medical 25:476–482, 1968. alcohol spectrum disorders. Genome Medicine 2(4):27, and iq. American Journal of Medical Genetics 47(6): li, e.; BesTor, T.h.; and JaenisCh, r. Targeted mutation of 2010. PMid: 20423530 857–861, 1993. PMid: 8279483 the dna methyltransferase gene results in embryonic lethality. Cell 69(6):915–926, 1992. PMid: 1606615 reik, W.; dean, W.; and WalTer, J. epigenetic reprogram- sulik, k.k. Critical periods for alcohol teratogenesis in ming in mammalian development. Science 293(5532): mice, with special reference to the gastrulation stage of liu, Y.; BalaraMan, Y.; Wang, g.; eT al. alcohol exposure 1089–1093, 2001. PMid: 11498579 embryogenesis. Ciba Foundation Symposium 105:124– alters dna methylation profiles in mouse embryos at 141, 1984. PMid: 6563984 early neurulation. Epigenetics 4(7):500–511, 2009. rodenhiser, d., and Mann, M. epigenetics and human dis- PMid: 20009564 ease: Translating basic biology into clinical applications. Tahiliani, M.; koh, k.P.; shen, Y.; eT al. Conversion of 5- CMAJ: Canadian Medical Association Journal 174(3): methylcytosine to 5-hydroxymethylcytosine in mam- MaY, P.a., and gossage, J. P. estimating the prevalence 341–348, 2006. PMid: 16446478 malian dna by Mll partner TeT1. Science 324(5929): of fetal alcohol syndrome. a summary. Alcohol Research 930–935, 2009. PMid: 19372391 & Health 25(3):159–167, 2001. PMid: 11810953 saMPson, P.d.; sTreissguTh, a.P.; BooksTein, F.l.; eT al. incidence of fetal alcohol syndrome and prevalence of TriMBle, k.C.; MolloY, a.M.; sCoTT, J.M.; and Weir, d.g. MaY, P.a.; gossage, J.P.; Marais, a.s.; eT al. The epidemi- alcohol-related neurodevelopmental disorder. Teratology The effect of ethanol on one-carbon metabolism: ology of fetal alcohol syndrome and partial Fas in a 56(5):317–326, 1997. PMid: 9451756 increased methionine catabolism and lipotrope methyl- south african community. Drug and Alcohol Dependence group wastage. Hepatology 18(4):984–989, 1993. saThYan, P.; golden, h.B.; and Miranda, r. C. Competing 88(2–3):259–271, 2007. PMid: 17127017 PMid: 7691709 interactions between micro-rnas determine neural pro- MCgiVern, r.F. low birthweight in rats induced by prena- genitor survival and proliferation after ethanol exposure: VeazeY, k.J.; Carnahan, M.n.; Muller, d.; eT al. alcohol- tal exposure to testosterone combined with alcohol, evidence from an ex vivo model of the fetal cerebral cor- induced epigenetic alterations to developmentally cru- pair-feeding, or stress. Teratology 40(4):335–338, tical neuroepithelium. Journal of Neuroscience 27(32): cial genes regulating neural stemness and differentia- 1989. PMid: 2814895 8546–8557, 2007. PMid: 17687032 tion. Alcoholism: Clinical and Experimental Research doi: 10.1111/acer.12080 [epub ahead of print], 2013. Miranda, r.C.; PieTrzYkoWski, a.z.; Tang, Y.; eT al. sChalinske, k.l., and nieMan, k.M. disruption of methyl PMid: 23488822 Micrornas: Master regulators of ethanol abuse and toxi- group metabolism by ethanol. Nutrition Reviews city? Alcoholism: Clinical and Experimental Research 63(11):387–391, 2005. PMid: 16370223 WaddingTon, C.h. The epigenotype. 1942. International 34(4):575–587, 2010. PMid: 20102566 Journal of Epidemiology 41(1):10–13, 2012. PMid: sChneider, r., and grossChedl, r. dynamics and interplay 22186258 o’learY, C. M. Fetal alcohol syndrome: diagnosis, epi- of nuclear architecture, genome organization, and gene demiology, and developmental outcomes. Journal of expression. Genes & Development 21(23):3027–3043, Wang, l.l.; zhang, z.; li, q.; eT al. ethanol exposure Paediatrics and Child Health 40(1-2):2–7, 2004. PMid: 2007. PMid: 18056419 induces differential microrna and target gene expres- 14717994 sion and teratogenic effects which can be suppressed shukla, s.d.; Velaquez, J.; FrenCh, s.W.; eT al. emerging by folic acid supplementation. Human Reproduction ogaWa, T.; kuWagaTa, M.; ruiz, J.; and zhou, F.C. role of epigenetics in the actions of alcohol. Alcoholism: 24(3):562–579, 2009. PMid: 19091803 differential teratogenic effect of alcohol on embryonic Clinical and Experimental Research 32(9):1525–1534, development between C57Bl/6 and dBa/2 mice: a new 2008. PMid: 18616668 Wang, x.; goMuTPuTra, P.; WolgeMuTh, d.J.; and Baxi, l.V. view. Alcoholism: Clinical and Experimental Research acute alcohol exposure induces apoptosis and increas- skinner, M.k. What is an epigenetic transgenerational 29(5):855–863, 2005. PMid: 15897731 es histone h3k9/18 acetylation in the mid-gestation phenotype? F3 or F2. Reproductive Toxicology 25(1): mouse lung. Reproductive Sciences 17(4):384–390, ooi, s.l., and henikoFF, s. germline histone dynamics 2–6, 2008. PMid: 17949945 and epigenetics. Current Opinion in Cell Biology 19(3): 2010. PMid: 20124552 257–265, 2007. PMid: 17467256 sMallWood, s.a., and kelseY, g. de novo dna methyla- Warren, k.r., and li, T.k. genetic polymorphisms: impact tion: a germ cell perspective. Trends in Genetics on the risk of fetal alcohol spectrum disorders. Birth PadManaBhan, r., and haMeed, M.s. effects of acute doses 28(1):33–42, 2012. PMid: 22019337 of ethanol administered at pre-implantation stages on Defects Research. Part A, Clinical and Molecular fetal development in the mouse. Drug and Alcohol sosCia, s.J.; Tong, M.; xu, x.J.; eT al. Chronic gestational Teratology 73(4):195–203, 2005. PMid: 15786496 Dependence 22(1–2):91–100, 1988. PMid: 3234238 exposure to ethanol causes insulin and igF resistance Wu, h.; d’alessio, a.C.; iTo, s.; eT al. genome-wide analy- and impairs acetylcholine homeostasis in the brain. sis of 5-hydroxymethylcytosine distribution reveals its Pal-Bhadra, M.; Bhadra, u.; JaCkson, d.e.; eT al. distinct Cellular and Molecular Life Sciences 63(17):2039– dual function in transcriptional regulation in mouse methylation patterns in histone h3 at lys-4 and lys-9 2056, 2006. PMid: 16909201 correlate with up- & down-regulation of genes by embryonic stem cells. Genes & Development 25(7): ethanol in hepatocytes. Life Sciences 81(12):979–987, sToCkard, C.r. The effect on the offspring of intoxicating 679–684, 2011. PMid: 21460036 2007. PMid: 17826801 the male parent and the transmission of the defects to Wu, s., and zhang, Y. active dna demethylation: Many subsequent generations. The American Naturalist roads lead to rome. Nature Reviews. Molecular Cell PandeY, s.C.; ugale, r.; zhang, h.; eT al. Brain chromatin 47(563):641–682, 1913. remodeling: a novel mechanism of alcoholism. Journal of Biology 11(9):607–620, 2010. PMid: 20683471 Neuroscience 28(14):3729–3737, 2008. PMid: 18385331 sToCkard, C.r., and PaPaniColaou, g. a further analysis of Yao, x.h., and nYoMBa, B.l. hepatic insulin resistance the hereditary transmission of degeneracy and defor- induced by prenatal alcohol exposure is associated with Park, P.h.; Miller, r.; and shukla, s.d. acetylation of histone maties by the descendants of alcoholized mammals. reduced PTen and TrB3 acetylation in adult rat off- h3 at lysine 9 by ethanol in rat hepatocytes. Biochemical The American Naturalist 50(590):65–88, 1916. and Biophysical Research Communications 306(2): spring. American Journal of Physiology. Regulatory, 501–504, 2003. PMid: 12804592 sTouder, C., and Paoloni-giaCoBino, a. Transgenerational Integrative and Comparative Physiology 294(6): effects of the endocrine disruptor vinclozolin on the r1797–1806, 2008. PMid: 18385463 Parnell, s.e.; o’learY-Moore, s.k.; godin, e.a., eT al. methylation pattern of imprinted genes in the mouse Magnetic resonance microscopy defines ethanol- zhong, l.; zhu, J.; lV, T.; eT al. ethanol and its metabo- sperm. Reproduction 139(2):373–379, 2010. PMid: induced brain abnormalities in prenatal mice: effects of lites induce histone lysine 9 acetylation and an alter- 19887539 acute insult on gestational day 8. Alcoholism: Clinical ation of the expression of heart development-related and Experimental Research 33(6):1001–1011, 2009. sTouder, C.; soMM, e.; and Paoloni-giaCoBino, a. Prenatal genes in cardiac progenitor cells. Cardiovascular PMid: 19302087 exposure to ethanol: a specific effect on the h19 gene Toxicology 10(4):268–274, 2010. PMid: 20811785

46 Alcohol Research: Current Reviews