Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Issue: Annals Meeting Reports

Behavioral

Barry M. Lester,1 Edward Tronick,2,3 Eric Nestler,4 Ted Abel,5 Barry Kosofsky,6 Christopher W. Kuzawa,7 Carmen J. Marsit,8 Ian Maze,9 Michael J. Meaney,10 Lisa M. Monteggia,11 Johannes M. H. M. Reul,12 David H. Skuse,13 J. David Sweatt,14 and Marcelo A. Wood15 1Departments of and and Pediatrics, Warren Alpert Medical School, Brown University, Women and Hospital, Providence, Rhode Island. 2Department of , University of Massachusetts, Boston, Massachusetts. 3Child Development Unit, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts. 4Mount Sinai Brain Institute, Department of , Mount Sinai School of Medicine, Fishberg Department of Neuroscience, Mount Sinai School of Medicine, New York, New York. 5Department of , University of Pennsylvania, Philadelphia, Pennsylvania. 6Divsion of Pediatric , New York-Presbyterian Hospital/Weill Cornell Medical Center, New York, New York. 7Institute for Policy Research, Northwestern University, Evanston, Illinois. 8Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island. 9The Rockefeller University, Laboratory of Chromatin Biology and Epigenetics, New York, New York. 10Departments of Psychiatry, Neurology, and , McGill University, Montreal, Quebec, Canada. 11Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, Texas. 12Henry Wellcome Laboratories for and Endocrinology, University of Bristol, Bristol, United Kingdom. 13Behavioural and Brain Sciences Unit, University College of London, Institute of Child Health, London, United Kingdom. 14Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama. 15Department of Neurobiology and Behavior, University of California, Irvine, California

Address for correspondence: Barry Lester, Ph.D., Brown Center for the Study of Children at Risk, Women and Infants Hospital, 101 Dudley St., Providence, RI 02908. [email protected]

Sponsored by the New York Academy of Sciences, the Warren Alpert Medical School of Brown University and the University of Massachusetts Boston, “Behavioral Epigenetics” was held on October 29–30, 2010 at the University of Massachusetts Boston Campus Center, Boston, Massachusetts. This meeting featured speakers and panel discussions exploring the emerging field of behavioral epigenetics, from basic biochemical and cellular mechanisms to the epige- netic modulation of normative development, developmental disorders, and . This report provides an overview of the research presented by leading scientists and lively discussion about the future of investigation at the behavioral epigenetic level.

Keywords: behavior; epigenetics; chromosome; regulation; transcription;

epigenetics. Behavioral epigenetics was described as Background and perspectives the application of the principles of epigenetics to What is behavioral epigenetics? the study of physiological, genetic, environmental, Barry M. Lester (Alpert Medical School of Brown and developmental mechanisms of behavior in hu- University) introduced the topic of the conference, man and nonhuman . Investigations typi- behavioral epigenetics, by describing research on the cally focus at the level of chemical changes, gene developmental origins of diseases, suggesting expression, and biological processes that underlie that the fetus is actually making adaptations through normal and abnormal behavior. This includes how programming to “prepare” for the postnatal en- behavior affects and is affected by epigenetic pro- vironment in response to environmental signals. cesses. Interdisciplinary in its approach, it draws These effects are due, in part, to epigenetic mech- on sciences, such as neuroscience, psychology and anisms, raising the fascinating question of whether psychiatry, , biochemistry, and psychophar- these mechanisms can also explain behavioral out- macology. Whereas there are thousands of studies of comes, thus providing an example of the kind of epigenetics that have been conducted over the last 40 research that could lead to a new field—behavioral years, the application of epigenetics to the study of

doi: 10.1111/j.1749-6632.2011.06037.x 14 Ann. N.Y. Acad. Sci. 1226 (2011) 14–33 c 2011 New York Academy of Sciences. Lester et al. Behavioral epigenetics

Figure 1. The figure shows the 96 articles on behavioral epigenetics grouped by the behavioral construct studied and the that were studied in each of the behavioral construct categories. behavior is just beginning. A literature search of ci- methylation), ATPase-containing protein com- tations found only 96 articles to date on behavioral plexes that move oligomers along a strand epigenetics (see Appendix). These articles were an- of DNA, methylation of DNA, and the binding of alyzed according to the behavioral construct that numerous transcription factors and transcriptional was studied (e.g., substance use, psychiatric ill- coactivators and , all of which act in ness, learning/memory, neurodevelopment, parent- a concerted fashion to determine the activity of a ing, , and neurodegenerative disorders), the given gene. Epigenetic regulation is crucial for ner- species studied (e.g., human, mouse, rat), the tissue vous system development. Specifically, it can help that was analyzed (e.g., brain, blood), the epigenetic elucidate how genes are affected by environmen- mechanisms that were studied (e.g., methylation, tal stimuli, including several common mental retar- histone modifications), and the particular genes in- dation syndromes and related neurodevelopmen- vestigated (Fig. 1). For example, in relation to par- tal disorders that are caused by abnormalities in enting, the most commonly studied genes were the chromatin-remodeling mechanisms. and FOS genes. The presen- Epigenetic regulation also occurs in the mature, tation concluded with cautionary notes about the fully differentiated brain and provides unique mech- unique issues involved in the study of behavioral anisms that may underlie the stable changes in epigenetics in . gene expression under both normal conditions (e.g., learning and memory) and in several pathological Epigenetics: basic processes and states (e.g., depression, drug , schizophre- mechanisms nia, and Huntington’s disease, among others). In Eric Nestler (Mount Sinai School of Medicine) pre- some rare cases (e.g., gene imprinting), epige- sented an overview of basic epigenetic processes netic modifications can be transmitted to offspring, , and mechanisms.1 2 A broad perspective of epige- which raises the possibility that behavioral experi- netics includes any structural adaptation in chro- ence in adult life might influence gene expression in mosomal regions that mediate altered rates of gene subsequent generations. However, there has not yet transcription. Epigenetic regulation, also known been definitive evidence for epigenetic transmission as chromatin remodeling, in , describes a of behavioral experience. While work on epigenetic process where the activity of a particular gene is mechanisms in the brain is still in early stages, it controlled by the structure of chromatin in that promises to improve our understanding of brain gene’s proximity (Fig. 2). Chromatin remodeling plasticity, the pathophysiology of neuropsychiatric is , involving multiple covalent modifica- disorders, and may lead to the development of fun- tions of (e.g., acetylation, phosphorylation, damentally new treatments for these conditions.

Ann. N.Y. Acad. Sci. 1226 (2011) 14–33 c 2011 New York Academy of Sciences. 15 Behavioral epigenetics Lester et al.

Figure 2. General scheme of chromatin remodeling. (A) DNA double helix wrapped around an octomer of histone proteins forming the unit of chromatin, the nucleosome. (B) Chromatin can be conceptualized as existing in two primary structural states: as active, or open, euchromatin in which histone acetylation opens up the nucleosome to allow binding of the basal transcriptional complex and other activators of transcription; or as inactive, or condensed, heterochromatin, where all gene activity is permanently silenced. In reality, chromatin exists in a continuum of several functional states (active, permissive, repressed, and inactivated). Enrichment of histone modifications, such as acetylation (A) and methylation (M) at histone N-terminal tails and related binding of coactivators (Co-Act) or (Rep), to chromatin modulates the transcriptional state of the nucleosome.

Epigenetics, intergenerational inertia, and that organisms must cope with everything from very human adaptation rapid and acute fluctuations (e.g., overnight fast Christopher W. Kuzawa (Northwestern University) followed by breakfast) to chronic conditions that explored the importance of the dynamic nature of change only gradually (e.g., ice ages or migrating to a epigenetic change as a means by which organisms new environment). A range of adaptive mechanisms adapt to environmental change.3,4 He emphasized allows human populations to adjust to these various

16 Ann. N.Y. Acad. Sci. 1226 (2011) 14–33 c 2011 New York Academy of Sciences. Lester et al. Behavioral epigenetics timescales of change (Fig. 3). Natural selection sifts ples that suggest an ability of the current generation through the gene pool to select gene variants well to entrain development to maternal characteristics suited to the most stable features of local ecologies. that reflect her cumulative experiences include the Rapid and reversible homeostatic processes lie at setting of growth rate to breast milk leptin the other extreme, maintaining internal constancy as a cue of maternal energetic history, and work against a backdrop of dynamic environmental con- in the Philippines suggesting that fetal growth may ditions, such as food intake or psychosocial stress. be calibrated to a woman’s cumulative nutritional He noted the adaptive importance of developmental experiences across her lifetime. In both examples, plasticity or the capacity grounded in epigenetic and offspring developmental biology is not sensitive to other changes that allows a single genome to create potentially transient (and thus unreliable) condi- a range of possible traits in interaction with the en- tions during the brief period of pregnancy or lacta- vironment (e.g., growing larger lungs when raised tion. Rather, the maternal resources and signals that at high altitude). Because organisms only develop are transferred to offspring may be more integrative once, changing development in response to envi- and cumulative in nature and, thus, potentially pro- ronmental conditions is generally an irreversible vide a more reliable basis for adaptive adjustment. process; thus, developmental plasticity is a suitable Kuzawa hypothesized that the timing of early sensi- mode of adaptation to environmental features that tive periods, during which many epigenetic settings are too chronic to be buffered by homeostasis, but are established, may be more than accidental, but that are also too transient for genetic adaptations to reflect the evolution of a conduit of sorts, allowing consolidate around. nongenomic information to be transferred between Kuzawa pointed out that many documented ex- generations. amples of epigenetic sensitivity involve the adoption Learning and memory of stable changes in gene regulation in response to experiences during limited, early stages of devel- The second session, moderated by J. David Sweatt opment (sensitive periods). Might it make adaptive (University of Alabama at Birmingham), served as sense for a long-lived species like humans to commit an overview of the roles for epigenetic mechanisms to a strategy for life so early in the life cycle? Lim- in long-term learning and memory processes and iting epigenetic sensitivity to early developmental highlights one of the most exciting contemporary windows may, in fact, create opportunities to ad- areas in the behavioral epigenetics field. The ses- just biology to more reliable environmental cues in sion was comprehensive in scope, covering cogni- the form of the mother’s own phenotype. Exam- tion and behavior, synaptic function and cellular

Figure 3. The timescales of human adaptability. Light gray, more rapidly responsive/less durable; black, slowest to respond/most durable. Epigenetic changes contribute to multiple modes of adaptation, including developmental and intergenerational processes that allow adjustment to gradual environmental change occurring on a decadal or multigenerational timescale. Modified after Kuzawa’s work.4

Ann. N.Y. Acad. Sci. 1226 (2011) 14–33 c 2011 New York Academy of Sciences. 17 Behavioral epigenetics Lester et al.

Figure 4. Schematic representation of epigenetic modifications. (A) In the nucleus, DNA coils and condenses around histones. Each octameric histone core contains two copies each of histones H2A, H2B, H3, and H4. The DNA–protein complex is referred to as chromatin. (B) The DNA-histone interaction occurs at the N-terminal tail of a histone, where, for example, on the H3 N-terminal tail, there are several sites for epigenetic marking via acetylation, methylation, and phosphorylation. (C) In and around gene promoters that are rich in cytosine-guanine nucleotides (CpG islands), methyl groups are transferred to CpG sites. This process, called DNA methylation, is catalyzed by a class of enzymes known as DNA methyltransferases. plasticity, biochemical signaling mechanisms, and hypothesis that DNA methylation marks can be molecular epigenetic mechanisms. The work de- modified in response to an organism’s experience scribed largely emphasized the specific epigenetic and that these marks play a role in dynamically reg- mechanisms of histone posttranslational modifica- ulating the gene transcription supporting synap- tion and DNA methylation. tic plasticity and long-term memory formation and maintenance (Fig. 4). Epigenetic mechanisms in memory formation Sweatt’s presentation also described several pieces Sweatt addressed the idea that conservation of epi- of evidence supporting the idea that DNA methy- genetic mechanisms for information storage rep- lation plays a role in memory function in the resents a unifying model in biology, with epige- adult central nervous system (CNS).6 Thus, he netic mechanisms being used for cellular memory at described how general inhibitors of DNA methyl- levels from behavioral memory to development to transferase (DNMT) activity alter DNA methyla- cellular differentiation.5,6 As background, Sweatt tion in the adult brain and alter the DNA methyla- discussed how DNA methylation and histone mod- tion status of the plasticity-promoting genes reelin ifications are the two most extensively investigated and bdnf. Additional studies demonstrated that de epigenetic mechanisms. As Sweatt described, until novo DNMT expression is upregulated in the adult recently it was thought that once laid down, these rat hippocampus after contextual fear conditioning epigenetic marks would remain unchanged for the and that blocking DNMT activity blocks contex- lifetime of the organism; recent studies, however, in- tual fear conditioning. In addition, results were pre- cluding those from the Sweatt laboratory, have chal- sented demonstrating that fear conditioning is as- lenged this view. Nevertheless, it is clear that DNA sociated with rapid methylation and transcriptional methylation and attendant changes in chromatin silencing of the memory suppressor gene protein structure are capable of self-regeneration and self- phosphatase 1 (PP1) and demethylation and tran- perpetuation, necessary characteristics for a stable scriptional activation of the plasticity gene reelin. molecular mark. Thus, Sweatt discussed the broad These findings have the surprising implication that

18 Ann. N.Y. Acad. Sci. 1226 (2011) 14–33 c 2011 New York Academy of Sciences. Lester et al. Behavioral epigenetics

Figure 5. HDAC3 modulates memory formation in a Nr4a2-dependent manner. (A) HDAC3-FLOX mice with dorsal hippocampal deletion of Hdac3 exhibit significantly enhanced long-term memory as compared to wild-type littermates (both groups received RISC free control). In contrast, siRNA-Nr4a2–treated HDAC3-FLOX mice exhibit no enhanced memory. (B) qRT-PCR shows that siRNA against Nr4a2 significantly reduces Nr4a2 expression in both HDAC3-FLOX and wild-type littermates. both active DNA methylation and active demethy- ing and memory are histone-modifying enzymes, lation might be involved in long-term memory con- especially histone acetyltransferases (HATs) and his- solidation in the adult CNS. tone deacetylases (HDACs) (Fig. 5). Finally, a recent series of studies were described5 In the first part of his talk, Wood presented his that found that the bdnf gene locus is also sub- lab’s research in examining the role of the CREB- ject to memory-associated changes in DNA methy- binding protein (CBP), a potent HAT and transcrip- lation, and, moreover, that this effect is regulated tional coactivator, in long-term memory. One lim- by the NMDA receptor. Data were also presented itation in studying the role of CBP in learning and indicating that neuronal DNMT-deficient animals memory has been the lack of genetically modified have deficits in contextual fear conditioning, the mice with sufficient spatial and temporal regulation. Morris maze learning task, and hippocampal long- The Wood lab used genetically modified CBP-FLOX term potentiation (LTP). Overall, Sweatt concluded mice, in combination with adeno-associated virus that DNA methylation is dynamically regulated in (AAV)–expressing Cre recombinase, to generate ho- the adult CNS in response to experience and that mozygous focal Cbp deletions in only area CA1 of this cellular mechanism is a crucial step in memory the dorsal hippocampus. This novel approach re- formation. sulted in the necessary spatial restriction to study the role of CBP in one brain region and its effect Chromatin-modifying enzymes in long-term on long-term memory; additionally, it provided the memory temporal restriction to study a homozygous deletion In the second presentation of the session, Marcelo of Cbp in adult mice, which avoids confounds from A. Wood (University of California, Irvine) dis- developmental or performance issues. The Wood cussed the role of chromatin-modifying enzymes in lab found that homozygous deletions of Cbp re- regulating gene expression required for long-term sulted in hippocampus-dependent long-term mem- memory formation. Why are chromatin-modifying ory impairments associated with decreased levels of enzymes needed to regulate gene expression? A sim- specific histone modifications and decreased gene plistic answer comes from the level that compaction expression.7 genomic DNA undergoes when being compressed In the second part of his talk, Wood presented to fit into a nucleus. Genomic DNA is two me- research examining the role of a specific HDAC in ters in length, yet has to fit into a six-micron nu- long-term memory formation. To date, the func- cleus, and, thus, must undergo an approximately tion of HDAC3, one of the most highly expressed 10,000-fold compaction. This generates an access class I HDACs in the brain, has never been ex- and indexing problem, which is solved in part by amined. Again, using AAV-expressing Cre recombi- chromatin-modifying enzymes. The best-studied nase and HDAC3-FLOX genetically modified mice, chromatin-modifying enzymes in the field of learn- the Wood lab demonstrated that HDAC3 is a key

Ann. N.Y. Acad. Sci. 1226 (2011) 14–33 c 2011 New York Academy of Sciences. 19 Behavioral epigenetics Lester et al. negative regulator of long-term memory forma- as a site of action for glucocorticoids in stress-related tion in the hippocampus. Homozygous deletion memory formation. of Hdac3 in area CA1 of the hippocampus led to In his presentation, Reul showed that the hor- enhanced long-term memory associated with in- mone’s action required distinct epigenetic modifi- creased levels of specific histone modifications and cations of the chromatin: the phosphorylation of increased gene expression. Similar results were ob- serine10 (S10), in combination with the acetylation served when an HDAC3 selective inhibitor, called of lysine14 (K14) of histone H3 (H3S10p-K14ac), RGFP136, was site specifically delivered to the dorsal leading to the induction of the immediate-early hippocampus. Together, the genetic and pharmaco- genes c-Fos and Egr-1 in dentate gyrus granule neu- logic data demonstrated that HDAC3 is a negative rons in rats and mice in vivo.11,12 As the gluco- regulator of long-term memory formation.8 corticoid receptor (GR) cannot bring about these In summary, Woodposited that HDACs represent histone modifications directly, Reul suggested that atypeofmolecularbrakepadthatisnormallyen- the GR acts indirectly via interaction with other sig- gaged but transiently removed by sufficient activity- naling molecules. More specifically, he postulated dependent signaling to regulate the transcription that GR interacts with the NMDA receptor-activated required for long-term memory formation.9 Wood ERK (extracellular signal-regulated kinase) MAPK concluded by suggesting that this process may rep- (mitogen-activated protein kinase) signal pathway, resent a molecular mechanism to explicate why we which has a marked role in learning and memory do not encode everything we experience into long- processes (Fig. 6; Refs. 12 and 13). Supporting this term memory. Moreover, impaired function of these notion, Reul presented unpublished in vivo data molecular brake pads may be associated with disor- clearly showing that in activated dentate granule ders including drug addiction and posttraumatic neurons, that is, those exhibiting phosphorylated stress disorder. ERK1/2 (pERK1/2), GRs are required to activate the downstream histone-modifying enzymes MSK1 Signaling and epigenetic mechanisms (mitogen and stress-activated kinase 1), and Elk-1 in stress-related memory formation (Ets-like protein-1) (Fig. 6; Refs. 12–14). In the final presentation, Johannes (Hans) Reul Reul went on to describe pMSK1, a kinase that (University of Bristol, UK) presented a novel mech- can phosphorylate histone H3 at serine10, whereas anism that may explain why we make such strong pElk-1 binds the HAT p300, which can acetylate his- memories of psychologically stressful and emotional tone tails. Further, showing a series of immunoflu- events in our lives. The mechanism he proposed orescence images, he demonstrated that, during the involves crosstalk between different signaling path- consolidation phase of memory formation, all par- ways influencing epigenetic processes in neurons of ticipating signaling molecules (pERK1/2, pMSK1, the hippocampus, a limbic brain region involved in pElk-1), modified histone molecules (H3S10p- learning and memory. Stressful events, for example, K14ac), and induced intermediate-early gene a domestic dispute or a job interview, or in ani- products (c-Fos, Egr-1) can be found in the same mals, an attack by a predator, evoke the secretion of dentate gyrus granule neurons.12,13 Furthermore, glucocorticoid hormones from the adrenal gland. he showed that blocking GRs led to a substan- Classically, these hormones regulate metabolic and tially decreased formation of pMSK1 and pElk-1, other physiological processes that enable an individ- but not pERK1/2, in dentate granule neurons af- ual to cope with the challenge in the best possible ter forced swim stress.12 He concluded that stress- way. ful events are strongly encoded into memory be- Reul reported, however, that research spanning cause of the marked activating role of GRs on ERK the last 25 years has provided evidence that gluco- MAPK, signaling to the chromatin in dentate gyrus corticoids secreted during a psychologically stressful neurons (Fig. 6; Ref. 12). These findings may be of challenge enhance the consolidation of memories significance for stress-related psychiatric disorders, related to the event—a long-standing observation such as major depression and anxiety, including that has remained unexplained until now. A finding PTSD. made in the 1980s by de Kloet’s group10 pointed to The formal presentations were followed by a the dentate gyrus, the gateway of the hippocampus, wide-ranging and lively discussion of the roles

20 Ann. N.Y. Acad. Sci. 1226 (2011) 14–33 c 2011 New York Academy of Sciences. Lester et al. Behavioral epigenetics

Figure 6. Glucocorticoid hormones, secreted as a result of a stressful event, enhance the consolidation of behavioral responses, including memories related to the event. Until recently, the underlying mechanisms of these effects were unknown. Recent work of the Reul group at the University of Bristol shows that glucocorticoids act by binding to glucocorticoid receptors (GRs) that interact with the NMDA receptor-activated ERK1/2/MSK1-Elk-1 signaling pathway enhancing the formation of epigenetic modifications (i.e., the serine10 phosphorylation and lysine14 acetylation in histone H3 (H3S10p-K14ac)) and the induction of the -associated immediate-early genes c-Fos and Egr-1 in sparsely distributed mature dentate gyrus neurons. Evidence has been accumulating that these signaling, epigenetic, and genomic phenomena are of critical importance for the consolidation of memories related to the endured stressful event. and regulation of epigenetic mechanisms in long- Alterations of DNA methylation, growth term synaptic plasticity and behavioral memory restriction, and infant neurobehavior in vivo. The first talk was by Carmen J. Marsit (Brown Uni- versity) and focused on altered epigenetic marks within the placenta. Epidemiological studies iden- Development tify variations in birth weight as a predictor of health This section, chaired by Edward Tronick (Univer- over the lifespan, including the risk for neuropsy- sity of Massachusetts Boston and Children’s Hospi- chiatric disorders.15 Marsit discussed a novel way tal Boston), focused on the emerging environmental in which to consider the effects of the intrauter- epigenetics hypothesis, which suggests that environ- ine environment on infant neurodevelopment in mental signals operate during early development to human populations, focusing on how differences alter epigenetic marks across the genome, thus in- in DNA methylation at specific genomic regions fluencing neural development and function. in the human placenta are associated with infant

Ann. N.Y. Acad. Sci. 1226 (2011) 14–33 c 2011 New York Academy of Sciences. 21 Behavioral epigenetics Lester et al. neurobehavior. The placenta acts as the master reg- to maternal (see Meaney presentation ulator of the intrauterine environment, not only below). through nutrients, gas, water, and waste exchange, Marsit also showed that these effects were most but also through the production of pregnancy- pronounced among infants of normal weight for related hormones, proteins, and growth factors, in- gestational age, suggesting that there may be normal cluding neuropeptide hormones analogous to those variability in placental methylation that accounts produced by the hypothalamus and pituitary. Fi- for variation in infant neurobehavior. As Marsit nally, the placenta also acts as a barrier commonly expands his studies of the role of the intrauterine metabolizing maternal hormones to inactive forms environment captured in the placenta , and, thus, stabilizing the intrauterine endocrine en- links between the methylation of key genes involved vironment. Such considerations have led to the con- in HPA axis control and infant neurobehavior, and cept that the placenta acts as the “third brain” by associations between genome-wide profiles of DNA linking the developed maternal physiological state methylation and infant neurodevelopment are being with the developing fetus. pursued. These studies are of particular importance Importantly, placental gene expression is subject as multiple environmental exposures, such as nutri- to environmental regulation. Marsit’s group con- ent deprivation, are known to affect infant growth sidered how changes to the patterns of DNA methy- and are associated with an increased risk for neu- lation in the placenta may alter the function of the rocognitive conditions, including deficit placenta in these critical roles and, in turn, how these hyperactivity disorder (ADHD). alterations manifest in neurobehavioral phenotypes in infants, characterized using the well-established Epigenetic alterations and exposure Neonatal Intensive Care Unit Network Neurobehav- to in utero ioral Scales (NNNS). Barry Kosofsky (Weill Cornell Medical College) dis- Marsit highlighted work linking patterns of DNA cussed how developmental brain disorders and the methylation in the placenta to the intrauterine en- consequences of prenatal exposure to drugs of abuse vironment represented by infant growth, showing (cocaine, in particular) are associated with sustained strong and significant associations between profiles changes in CNS gene expression and have last- of DNA methylation, identified using genome-wide ing consequences for brain structure and function array-based approaches, and infant birth weight.16 (Fig. 8).17 Prenatal exposure to toxins, including He went on to demonstrate that increasing methy- substances of abuse, is associated with developmen- lation of the human GR 1F was strongly and sig- tal effects in children. Kosofsky suggested that such nificantly associated with decreased measures of aberrant effects might be considered “molecular infant attention on the NNNS (Fig. 7). Impor- malformations,” leading to conditions where neural tantly, the methylation of an analogous receptor signaling pathways are rendered dysfunctional. Such (rat exon 17) in rat pup hippocampus has linked molecular changes may “feed forward” to produce

Figure 7. Association between greater than median glucocorticoid receptor exon 1F methylation and infant attention score is specific to nongrowth restricted infants.

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Figure 8. Epigenetic mechanisms underlying persistent alterations on promoters of genes involved in neuronal plasticity. alterations in the behavioral repertoire of affected demonstrating that mice exposed to cocaine pre- infants, children, and young —changes that natally demonstrated impaired spontaneous recov- are sculpted by environmental interactions. Kosof- ery of extinction, a form of learning that relies on sky’s research explores the hypothesis that the result- the mPFC. The prenatally cocaine-exposed animals ing molecular maladaptations are, in part, mediated showed a decrease in the binding of MeCP2 to the by epigenetic mechanisms. of exons I and IV of the bdnf gene, which Kosofsky presented data from a mouse model was associated with decreased mRNA expression of transplacental cocaine exposure. These findings of those transcripts in mPFC, likewise suggesting suggest that changes are expressed in a gene-specific, an epigenetic mechanism underlying the behavioral region-specific, and time-specific fashion; when ap- alterations. These findings are consistent with the parent in the medial prefrontal cortex (mPFC), these presentation of David Sweatt in an earlier session changes appear to result in altered structural and on epigenetic mechanisms for learning and mem- functional maturation of that brain region. When ory that highlighted the importance of epigenetic compared with control animals (i.e., mice with no regulation of the bdnf gene for fear conditioning. prenatal drug exposure), the cocaine-exposed mice The implication from these findings is that perina- showed a differential pattern of performance on a tal environmental conditions might determine the social interaction (SI) task: increased SI relative to capacity for neural plasticity in later life through controls at P28 (juvenile) and decreased SI relative epigenetic regulation of genes critical for synaptic to controls as adults. A parallel pattern of expres- remodeling. Kosofsky’s group is currently pursuing sion of the mRNA for the EGR1 “rescue experiments” to further explore the link be- (also known as NGF-1a and zif 268) was observed in tween the proposed molecular mechanisms in the mPFC corresponding with these ages. In adult an- animals prenatally treated with cocaine. The find- imals, changes in EGR1 expression correlated with ings may provide an opportunity for translational decreased binding of MeCP2 to the EGR1 promoter; benefit regarding the diagnosis and treatment for the same pattern was not observed at P28. Vari- the offspring of woman who abuse drugs during ations in MeCP2 occupancy suggest that an epi- pregnancy. genetic mechanism may underlie changes in gene expression and behavior. Epigenetic programming by maternal care Kosofsky presented additional behavioral studies Michael J. Meaney (McGill University) summarized using an “extinction of conditioned fear” model, previous studies showing that variations in maternal

Ann. N.Y. Acad. Sci. 1226 (2011) 14–33 c 2011 New York Academy of Sciences. 23 Behavioral epigenetics Lester et al.

Figure 9. Tactile stimulation derived from pup LG increases 5-HT activity at the level of the hippocampus, thus increasing NGFI-A expression and its association with the exon 17 promoter, which then initiates an alteration of the methylation state of the exon 17 glucocorticoid receptor promoter. care in the rat, specifically in the frequency of pup mone. T3 increases central 5-HT activity in the rat licking/grooming (LG), is associated with increased pup, and its administration is sufficient to increase methylation of the exon 17 GR promoter in the the association of NGFI-A with the exon 17 pro- hippocampus, decreased hippocampal FR expres- moter. Pup LG from the mother directly increases sion, and increased hypothalamic-pituitary-adrenal NGFI-A association with the exon 17 promoter, and (HPA) responses to stress.18,19 Previous work sug- artificial tactile stimulation mimics this effect. These gests that reversing the effects of differential DNA findings suggest that the tactile stimulation derived methylation of the exon 17 promoter, in turn, can from pup LG increases 5-HT activity at the level of reverse the effects of maternal care on hippocampal the hippocampus, thus increasing NGFI-A expres- FR expression and HPA responses to stress. Meaney sion and its association with the exon 17 promoter, also presented findings from studies in the post- which then initiates an alteration of the methyla- mortem human hippocampus showing that a de- tion state of the exon 17 GR promoter (Fig. 9). velopmental history of child abuse was associated The results are consistent with previous studies in with an increase in the methylation of the exon 1F vitro showing that overexpression of NGFI-A alters GR promoter (also see Marsit summary) and de- the methylation of the exon 17 promoter. Interest- creased GR expression. The focus of the talk con- ingly, NGFI-A also regulates the expression of the cerned the mechanisms by which the environmen- GAD1 gene that encodes decaroxy- tal signal, pup LG, might generate the difference lase 1, and maternal care regulates the methylation in DNA methylation, and gene expression. Meaney of the GAD1 and GAD1 expression in a manner summarized in vitro and in vivo evidence for the im- comparable to that of the GR. These studies are portance of serotonin (5-HT)- and 5-HT–induced consistent with earlier reports of alterations in DNA increases in hippocampal NGFI-A expression for the methylation associated with increased transcription alterations in the methylation state of the exon 17 factor binding, and suggest that environmental con- promoter. A shRNA targeting NGFI-A blocks both ditions can directly alter epigenetic states through the effects of 5-HT on the methylation state of the the activation of intracellular signaling pathways. exon 17 promoter and effects on GR expression. Pup Meaney also noted important caveats, most notably, LG provides tactile stimulation of the pup, resulting the importance of identifying the enzyme directly in an increase in circulating levels of triiodoithyro- responsible for the alteration in the methylation nine (T3), the most biologically active thyroid hor- state.

24 Ann. N.Y. Acad. Sci. 1226 (2011) 14–33 c 2011 New York Academy of Sciences. Lester et al. Behavioral epigenetics

Summary of symposium on development nisms by which drugs of abuse and stress produce Each of these presentations focused on well- long-lasting alterations in gene expression and be- established environmental influences, including havior.20 Moreover, identifying common regulatory pre- and postnatal maternal effects and drugs of mechanisms in cocaine and stress models may aid in exposure. This research reflects an emerging body the development of therapeutics aimed at alleviating of science examining epigenetic states as candidate addiction and depression syndromes. mechanisms that link environmental conditions in Epigenetic targets in neurodegenerative and early development with sustained changes in gene psychiatric disorders expression and neural development. Predictably, the Ted Abel (University of Pennsylvania) focused discussion focused on the enthusiasm for the po- on another type of crucial histone modification, tential benefits of interventions targeting epigenetic namely, histone acetylation, which is generally as- mechanisms. The speakers noted that the current sociated with transcriptional activation. Histone period marks a very early stage for research linking acetylation is catalyzed by HATs and reversed by environmental conditions to alterations in gene ex- HDACs. One of the major HATs present in the brain pression and brain function. Nevertheless, the trans- is the transcriptional coactivator, termed CBP. Abel lational science presented within this symposium et al. have shown that CBP is involved in synaptic suggests that epigenetics represents a fruitful area plasticity in the hippocampus and in specific forms of research bridging epidemiological findings with of long-term memory mediated via hippocampal studies of biological mechanism. circuits. Thus, mutant mice, in which CBP activ- ity in neurons is reduced, exhibit deficits in spatial and contextual memory and in long-lasting forms Histone methylation in cocaine-induced of hippocampal synaptic plasticity. A complemen- behavioral and structural plasticity tary method to study the role of histone acetylation Ian Maze presented research from Eric Nestler’s lab- in synaptic plasticity and memory is to examine the oratory (Mount Sinai School of Medicine) that di- effects of HDAC inhibitors, which increase histone rectly implicates a role for repressive histone methy- acetylation and transcriptional activation. The Abel lation, specifically dimethylation of Lys9 on histone laboratory and other groups have found that ad- H3 (), in cocaine addiction (Fig. 10). ministration of an HDAC inhibitor, such as tricho- Maze et al. have shown that chronic cocaine statin A, enhances long-term contextual memory administration to mice reduces global levels of and facilitates synaptic plasticity via the transcrip- H3K9me2 in (NAc), a key brain tion factor CREB. Among important target genes region involved in processing reward and implicated induced by HDAC inhibition and CREB in the hip- in addiction.20 InNAc,thereductioninH3K9me2 pocampus are certain nuclear receptor transcription is mediated through decreased expression of G9a, a factors that are critical for the enhanced cognitive histone methyltransferase that specifically catalyzes ability observed. Histone acetylation may, therefore, H3K9me2. The repression of G9a, in turn, is me- provide an epigenetic mechanism for establishing diated by the cocaine-induced transcription factor, gene-specific modifications that result in the co- FosB. Using conditional mutagenesis and viral- ordinate expression of genes required for long-term mediated gene transfer, the group found that G9a memory storage. As well, HDAC inhibitors may pro- downregulation increases dendritic spine plasticity vide a novel therapeutic approach to treat the cogni- of NAc neurons and enhances rewarding responses tive deficits that accompany many neuropsychiatric to cocaine by decreasing repressive H3K9me2 at spe- disorders. cific target genes and, therefore, increasing those genes’ expression. Cocaine-induced downregula- Epigenetic mechanisms regulating tion of G9a and H3K9me2 also promotes an in- function and behavior dividual’s vulnerability to stressful experiences and Lisa M. Monteggia (The University of Texas South- the development of depression-like behavioral ab- western Medical Center at Dallas) discussed her lab- normalities. These findings are consistent with clin- oratory’s studies of loss-of-function mutations in ical data that drug addiction and depression often the gene methyl-CpG-binding protein-2 (MeCP2) occur together. This work has defined new mecha- that cause Rett syndrome, a neurodevelopmental

Ann. N.Y. Acad. Sci. 1226 (2011) 14–33 c 2011 New York Academy of Sciences. 25 Behavioral epigenetics Lester et al.

Figure 10. Mechanism of increased cocaine sensitivity. disorder characterized by reduced cognitive func- tion of synaptic function. The regulation of these tion and autism spectrum-like behavioral abnor- two key epigenetic mechanisms by synaptic activity, malities, among other deficits. and how such alterations affect , Monteggia et al., along with other groups, have will be critical to elucidate the mechanisms under- demonstrated that mice lacking MeCP2 exhibit ab- lying Rett syndrome as well as the roles these factors normal cognitive and social behavior. The group have in basic cellular processes. This work is also has also demonstrated that loss of MeCP2 de- essential in understanding abnormalities in neuro- creases excitatory glutamatergic transmission in transmission that underlie Rett syndrome and other the hippocampus, primarily by reducing glutamate neuropsychiatric disorders. release. By contrast, no deficit in inhibitory GABAergic function is seen. The results suggest Epigenetic risk factors in that some Rett abnormalities are caused by an social-communication disorders imbalance between excitatory and inhibitory neu- David H. Skuse (University College London in the rotransmission in particular brain circuits, a possi- UK) discussed genomic imprinting, which involves bility supported by work from the Monteggia lab- epigenetic modifications that result in differential oratory. MeCP2, encoded by the X chromosome, gene expression from certain genes (or even entire functions predominantly by binding to methylated chromosomes) that are of paternal versus maternal CpG islands in the promoter region of certain genes origin. and thereby silencing those genes’ expression. This Importantly, Skuse et al. have proposed that occurs, in part, by forming a protein complex with imprinting of the X-chromosome could result in HDACs, which also repress gene activation, as noted sexually dimorphic characteristics. This hypothesis above. The Monteggia laboratory has, therefore, predicts that sexually dimorphic (male) vulnerabil- started to investigate the coordinated role of histone ity to some neurodevelopmental disorders, such as deacetylation and DNA methylation in the regula- autism, could occur on the basis of whether the

26 Ann. N.Y. Acad. Sci. 1226 (2011) 14–33 c 2011 New York Academy of Sciences. Lester et al. Behavioral epigenetics silencing of alleles is confined to chromosomes that ified. Yet any or all of these environmental factors, were either paternally or maternally derived.21 and the animals’ engagement with them, may lead The X-chromosome is enriched for genes that to epigenetic effects. Indeed the presumption of epi- are involved in brain function. X-monosomy in hu- genetics is that such factors do and will have effects. mans results in Turner syndrome, which provides a For example, the stability of epigenetic changes may model by which these putative mechanisms can be be the result of changes in environmental factors studied. In general, studies have shown that females that function to stabilize, destabilize, or even reit- with a single X-chromosome of paternal origin have eratively induce epigenetic changes rather than the better social communication skills, and are more stability being “inherent” to the epigenetic change empathic, than those whose single X-chromosome in and of itself. Moreover, the dynamics of change is maternal in origin.22 Autosomal gene expression need to be better understood; that is, epigenetic may be regulated by X-linked genes.23 changes related specifically to external environmen- Skuse’s laboratory has shown in longitudinal tal events in turn can become causal elements that go studies that these differences persist from childhood on to amplify, stabilize, or inhibit other epigenetic into adulthood. These human observations have changes. These dynamics have come to be appreci- more recently been followed-up by studies of X- ated in studies of physiologic systems such as the monosomic mice. Replicated findings include pref- HPA axis, where physiologic and behavioral feed- erential expression of alleles from the maternally back and feed-forward loops operating over time are derived X-chromosome. One particular allele, in- critical to understanding how the organism func- variably expressed in males, is associated with per- tions. Similar dynamic thinking needs to be intro- severative behavior.24 No evidence for preferential duced into our models of epigenetic changes. expression of the paternally derived X-chromosome Tronick noted that most of the models we have has yet been observed, although recent work from for epigenetic mechanisms related to behavior are Catherine Dulac’s laboratory at Harvard has pro- models of abnormal processes, such as toxic expo- vided support for the role of X-linked imprinting in sures or deprivation. We know less about epigenetic brain development.25 processes that affect normal behavior as was shown Better understanding the mechanisms of genomic in work of Marsit. Thus much of what we know imprinting has the potential of providing new in- may be related to aberrant processes that fall out- sight into the molecular basis of individual variabil- side the range of normal epigenetic processes. For ity in traits as well as features of neu- example, the finding of high levels of methylation ropsychiatric disorders. early in development could suggest that the timing of the effects of experience may be critical to under- Concluding Remarks standing epigenetic effects on behavior. Moreover, This conference brought together cutting-edge an- the idea of developmental change as being, in part, a imal and human research in the emerging field of process related to a release from methylation would behavioral epigenetics. As exciting as these findings have profound effects for our understanding of de- are, there are a number of challenges that need to velopment, such as identifying the events and their be addressed as the field moves forward. Ed Tronick timing that trigger the release from methylation, as (University of Massachusetts and Harvard Medical well as for therapeutic interventions. School) pointed out that given what is now known Tronick recognized that at the present time our about environmental effects that there needs to be ability to specify the chain of causality of epigenetic as much effort put into characterizing the details of changes in human behavior is limited because of our the experience of the or human and its envi- inability to access brain tissue. One can only hope ronment as has gone into characterizing molecular thatnewtechniqueswillbedevelopedthatovercome mechanisms. At the moment experiential and envi- this limitation and that with a better understand- ronmental “phenotyping” is crude. Even in the best ing of tissue may lead to the finding of “substitutes animal experimental studies, factors in addition to tissue” and correlated changes in other physiologi- the study proper, such as housing conditions, events cal systems. For example, epigenetic changes in the during animal housing, handling regimes, light cy- glucocorticoid receptor detected in plasma or buc- cles, social contacts, to name a few are not well spec- cal cells accompanied by parallel changes in ACTH,

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CRF or cortisol would strengthen the role of the HPA References axis. But while we wait for these new innovations 1. Jenuwein, T. & C.D. Allis. 2001. Translating the histone code. there needs to be an appreciation of the limitations Science 293: 1074–1080. of human epigenetic work compared to the research 2. Tsankova, N., W. Renthal, A. Kumar & E.J. Nestler. 2007. done on animals. The state of the art of the two areas Epigenetic regulation in psychiatric disorders. Nature Rev. is not the same, and applying state-of-the-art animal Neurosci. 8: 355–367. standards to human work will only limit progress. 3. Kuzawa, C.W.& E.A. Quinn. 2009. Developmental origins of adult function and health: evolutionary hypotheses. Annu. Tracing human behavior to epigenetic changes will Rev. Anthropol. 38: 131–147. bedifficult,butwecanmakeeveryefforttohave 4. Kuzawa, C.W. 2005. 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Apr. 20 [Epub ahead of print]. behavioral scientists, who are capable of developing 8. McQuown, S.C., R.M. Barrett, D.P. Matheos, et al. 2011. models that will elucidate both the hidden links and HDAC3 is a critical negative regulator of long-term memory the complexity to further advance this relatively new formation. J. Neuorsci. 31: 764–774. field of behavioral epigenetics. 9. McQuown, S.C. & M.A. Wood.2011. HDAC3 and the molec- ular brake pad hypothesis. Learn. Mem.Apr.16[Epubahead of print]. Acknowledgments 10. De Kloet, E.R., S. De Kock, V. Schild & H.D. Veldhuis. 1988. Antiglucocorticoid RU 38486 attenuates retention of a be- The Behavioral Epigenetics conference was pre- haviour and disinhibits the hypothalamic-pituitary adrenal sented by the New York Academy of Sciences, the axis at different brain sites. Neuroendocrinol. 47: 109–115. Warren Alpert Medical School of Brown Univer- 11. Bilang-Bleuel, A. et al. 2005. increases sity, and the University of Massachusetts Boston, histone H3 phosphorylation in adult dentate gyrus gran- ule neurons: involvement in a glucocorticoid receptor- and supported in part by the University of Mas- 22: TM dependent behavioural response. Eur. J. Neurosci. 1691– sachusetts Boston and the Life Technologies 1700. Foundation (Silver), the Massachusetts Life Sciences 12. Gutierrez-Mecinas, M., A. Collins, X. Qian, et al. 2009. Center (Bronze), and Bristol-Myers Squibb R&D Forced swimming-evoked histone H3 phospho-acetylation and Genomatix Software, Inc (Academy Friends). and c-Fos induction in dentate gyrus granule neurons in- Funding for this conference was also made possi- volves ERK1/2-mediated MSK1 and Elk-1 phosphorylation. Soc. Neurosci. Abst. 777: 17. ble by (i) Grant 1 R13 DA029985-01 from the Na- 13. Reul, J.M.H.M., S.A. Hesketh, A. Collins & M. Gutierrez- tional Institute on Drug Abuse, the Eunice Kennedy Mecinas. 2009. Epigenetic mechanisms in the dentate gyrus Shriver National Institute of Child Health and Hu- act as a molecular switch in hippocampus-associated mem- man Development, the National Institute of Mental ory function. Epigenetics 4: 434–439. Health, and the National Institutes of Health, Office 14. Chandramohan,Y.,S.K. Droste, J.S. Arthur & J.M.H.M. Reul. 2008. The forced swimming-induced behavioural immobil- of the Director (Barry M. Lester, Principal Investiga- ity response involves histone H3 phospho-acetylation and tor); (ii) an Independent Medical Education Grant c-Fos induction in dentate gyrus granule neurons via ac- from AstraZeneca; (iii) March of Dimes Founda- tivation of the N-methyl-D-aspartate/extracellular signal- tion Grant No. 4-FY10-458; and (iv) an educational regulated kinase/mitogen- and stress-activated kinase sig- 27: grant from Janssen, a division of Ortho-McNeil- nalling pathway. Eur. J. Neurosci. 2701–2713. 15. Schlotz, W. & D.I. Phillips. 2009. Fetal origins of mental Janssen Pharmaceuticals, Inc., administered by health: evidence and mechanisms. Brain Behav. Immun. 23: Ortho-McNeil-Janssen Scientific Affairs, LLC. 905–916. 16. Filiberto, A.C. et al. 2011. Birthweight is associated with Conflicts of interest DNA promoter methylation of the glucocorticoid receptor in human placenta. Epigenetics May 1; 6(5). [Epub ahead of The author declares no conflicts of interest. print].

28 Ann. N.Y. Acad. Sci. 1226 (2011) 14–33 c 2011 New York Academy of Sciences. Lester et al. Behavioral epigenetics

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