oh i” @ Fron tHe acavem: couoquium inTRoDUCTION ® GrossMark Epigenetic changes in the developing brain: Effects on behavior Eric B. Keverne*, Donald W. Pfaff*, and inna Tabansky”™* “Sub-Department of Animal Behaviour, University of Cambridge, Cambridge CB3 BAA England; and "Laboratory of Neurobiology and Behaviour The Rockefeller Univesity, New York, NY 10021 “This Sackler Colloquism encompasses bread range of topics for the folowing reason. Our knowledge of the chemistry of epigenetic modifications is expanding at a rapid rate, but most ofthe primary discoveries in this filé are made using nonneural tissue. So, neuroscientists want to lear about this chem istry but may not have direct exposure to the materi In a complementary fashion, molecular geneticists and protein chemists who experiment on DNA methylation, histone modifications and noncoding RNAS realize that some ofthe most exciting applications of their discovers are in the CNS, but for such scientists behavioral assays for example, are distant from their expertise. The purpose of this Sackler Colloguium, Uherefore, was to bring together experts in the two fields— epigenetic chemistry and behavioral neuro science in the sprit of mutual education. ‘We start with the chemistry of transcription ise, Decades of biochemical work led to the concept ofthe bas transcriptional complex (0,2), and recently a structure for the tran- sctiption preiniation complex was report (9) Neurassentsts concemed with the deve- ‘opment of behavior need to know the varity ‘of changes in the cll leu tht precede the formation ofthat comples, with an emphasis ‘on those meaifcations that ls lang. time It has been established that specific genes ‘operating in specific neurons govern the appearance of specific, biologically crucial Ichaviors. The system that has beer worked ‘oat in the most detail features estrogen development. Its contribution to placentation, metabolism, thermogenesis (body temperature regulation), maternal care, growth, and brain development are global functions targeted by imprinted ‘gene regulation, Genomic imprinting is regulated by epi- ‘genetic marks in the imprint control region OCR) of genes. These marks are heritable and reslt in the monoallelic, parent of ori- {gin-dependent expression of genes. The ICRs are primarily maternally methylated and are inherited and reprogrammed through the matriline (28), with three main exceptions (H19, RU, and DIK). Imprinting of reto- transposon-Like 1 (Rif) and delta-Like 1 homolog (Dik) genomic regions is thought to have occurred afer the marsupial-cuthe- rian divergence, with the accumulation of ‘miRNAs and SoRNAS on the matemal chromosome, whic hasan active role in the regulatory expression of these genes (29) The ICRS have gained regulatory control ‘over clusters of genes increasing in number from marsupials to rodents to humans, and ‘of particular importance for tight regulation ‘over gene dosage. The significance of this regulation is illustrated through the com- plexity of disrupted phenotypes that occur with perturbations to the ICR without struc tural mutations in the genes themsehes (- amples seen in Angela's syndrome, Prader Will syndrome, and Siver Rasel syndrome) (60). eis important to emphasize this nota singe gene effect and many imprinted genes are coregulated as part of an imprinting network that serves a similar global func- tion (growth, metabolism, maternal care) (31) In addition, each imprinted gene network may itself regulate a network of downstream {genes engaged in specific celular Functions (62) (development of pluripotent eels that ‘become committed to produce and respond to signals as part of global tissue function). or example, the paternally expressed 3 (eg3) transcriptome has a network of 22 ‘genes concerned with neural development, and 21 genes that are roulting other tran” scription factors, Peg aso regulates a number ‘of genes concerned with. placental deve- lopment (pregnaney specific glycoproteins, czacams, and prolatng), These genes have undergone multiple duplications across mammalian species (Pys' 1-28, Ceas 1-19, Pris 3-18) with the lowest copy number in rmetatherians and highest in eutherian primates. An important feature of genes that are imprinted is ther evolutionary stability through pufvng seen, andthe erstment ‘of mote genes to this epigenctic mode of regulation (ICRs) has occurted across the evolution of mammalian species (33). This recruitment has often expanded the ICR to include noncoding RNAS. Imprinted gene cluster contain atleast ane long noncoding RNA (lncRNA), and two clusters in patic~ ular (Kengl-Kengl oth; IgP-Aim) have an ‘timated 100 kb of hacRNA transcribed ‘an anisense direction from within Ue proten- ‘ning gene (3). The IncRNA plays an es- sential ole inthe imprinting ofthese clusters and deletion oftheir promoter rests in bak lalicexpresion. The imprinted genes are themselves re ‘markably stable and have not undergone subsequent gene duplication, nor is there evidence for positive selection of the pro- tein-coding genes in placental species (35). “The evolatonary stability of imprinted genes and robustness oftheir fanctional networks means that interacting hubs can prove for compensatory actions through common dovesteam genes Thus, minor enor can be absorbed and robustness preva inthe fanc- tionally complex biological systems they de- velop. Such error avoidance is essential for imprinted genes that are intergenerationally coexpressed in development (placenta), the interactions of which are of fandamental significance tothe survival ofboth generations ‘Many of the downstecam genes Uhl are regulate in cis by imprinted genes are them- selves monoallsically expresed. ENCODE, data (36), identified a subnetwork of rono- allelialy expressed genes that comprised rmateral or paternal specific networks. Within cach “alee effect network” those genes 1e- uated by increasing numbers of transcription factors tend to he Under stronger negative 8 lection, whereas genes tolerant of loss-of functions are under weaker negative slection ‘Genomic imprinting has ths become a co Gulinator of coadeyted gone expression. ‘iewpoint that has recently boen given song theoretical support (37), These kinds of net ‘work interaction often favor the evhiton of genetic coadaptaton where beneficaly inter cing alles evolve to become coher. Histone Acetylation and Methylation. In ‘genomic DNA, nucleosomes comprising ~146 ‘nucleotide bases tightly associated with his tone proteins are linked to reduced ease of Kevere ataoh i” transcription, Covalent modifications of the Netermini of histones haven ‘been hypothesized (38) to form an epigenetic histone code” that is ead by other nuclear proteins to facitate or repress transcription ‘of nearby genes, ‘Ths hitone variants can be “writen” “read” and “erased” as epi genetic marks during neural development. Although the study or histone variants and their posttranslational maxlifcations have largely been pursued in nonneuraltsue, certs 3.3 mutations have been reported 2s highly specific to pediatric gliomas (39), and histone modifications were drawa ito neural development during this meeting That inthis uc of PNAS Noh cal (40) repor the “reading” of a combinatorial his tone modification, HKSme3810ph in post mitotic neurons and relate i to periods of hsightened activity. Retrotransposons.Rtrotranspsons ae sl ith genetic ements that may be used to drive ‘evolution, but which may also rate serious ‘eosin neural development (Ret syndrome) and somatic development (cnc) The evo- lutionary impact of retrotransposons has depended on genetic mechanisms to control thee stability (1, "The epigenetic inheritance ‘of rtotransposon conta over gene expres sion probably ects ther predisposition for DNA methylation, Trim28 is a key player in silencing of endogenous retetransposons (42), and together with ZEPS7 controls levels ‘of methylation saeguarding the transcriptional dynamics of early embryos The resulting resistance of retrotranspons to reprogramming leads not only to trans ‘generational epigenetic elects, but in some ass to the parent of origin effect sen in {genomic imprinting 43). ft has been iste thatthe origins of genomic imprint- ing were based on retrtransposon insertion, though to date this has been shown to be the case only in one imprinted gene [Ras protein specific guanine nucleotide releasing factor 1 RasgyD}]-Imprintng ofthe Raserft Tocus depends on a noncoding RNA and PIWI interacting RNA (piRNA). A retro: transposon within the RNA is targeted by PIRNAS, which directs the sequence specific methylation of Rasgr (44). Recent evi dence indicates that microRNAs were ini tally formed from transposable element sequences (45). Retrotransposition has also resulted in the imprinting of other genes that ae imprinted ppSp, Nop 15, Mei2, and U2afl-rs), which are expressed in the brain (4). “Molecular mechanisms whereby trans {generational inheritance of transposons re sults in changes to DNA methylation that Kevere et extend into closely located epileles have been proposed to result in transgenerational transmission in plants (47). A number of studies in mammals have also found an as sociation between phenotypes and silencing of transposable elements, suchas that which jccurs in the Agouti mouse. In these ani ‘mals, transcription resulting from a retro transposon insertion 100-Kb upstream of the Agouti gene causes ectopic expression of this gene, leading to yellow fur, obesity, and diahetes (48). The distribution of yellow-fur phenotype differs according to a maternal epigenetic effect, not resulting from the en vironment but because of incomplete era- sure of epigenetic modification when a silenced allele is passed through the female bt not male geem line. ‘The coat color of ‘Agouti mouse progeny can be modulated by a diet rch in “methyl donors” but this i lost by the third generation, and is nether stable nor transgenerational (49). However, this same genetic locus has been shown to be under rapid adaptive selection for coat color, raising the possibly that some haplotypes may be prone to epigenetic variation (50) Elevation of L1 transposons is thought to induce meiotic prophase | defets, including cocyte ancuploidy and potential embryonic lethality (51, 52). 1 is well established that attrition of more than 60% of oocytes in meiotic prophase occurs before birth (53- 58). Fetal oocyte attrition may therefore serve to select “healthy” oocytes with limited retroransposon activities tht ae best sited for normal development of the next gener tion (9). ‘he brain isan important target for et rotransposon mobilization. The linel pro- ‘moter has binding sites for the YY1 and sex- determining region Y-box 2 (Sox2) neural transcription factors. Linel sequences are usually transcriptionally repressed asa re sult of their methylation in primordial germ cells, However, Mtr et al (60) reported LL RNAs to be present in neural progenitor calls derived from the hippocampus, which is congruent with the findings that LI is hypomethyated in the developing bran, Li expression has been shown to be repressed in neural stem cells by expression of Sox2 transcription factor and transcriptionally silened by DNA methylation, Recent studs have shown that neuronal progenitor cells derived from tissue of patients with Ret syndrome and earying methy/-CpG-binging protein (MeC?p2) mutations have increased. susceptibility to LI retrotransposition (61) Coufal etal. (62) found high levels of MeCp2 sociated with the L1 promoter modulating its developmental activity, but the DNA methyl binding protein 1 did not affect LT retrotransposition. LI insertions may be pre sent in only a subset of neurons, thereby producing L1 mosticism in the brain, Be cause of L1 mobilization in the later phases of Reurogenesis, each neuron may be genetically ‘unique with regard tothe cohort af loi con taining somatic LI insertions Ths expecially Pertinent to the hippocampus, where neuro: genesis continues from the subventricular zone into adulthood, Such mosaicisms ean produce genetic vesty among subpopulations of neurons. Importantly, LINEI germ-line insertions are rarely found in regions where they generate a deleterious phenotype be- cause of strong section against such mutations uring evolution. However, the mistegulation fof mobile elements has been found in the neural disorders of Rett syndrome and schizophrenia (63). Epigenetic Reprogramming of Neurons. The primorcal germ ell of the Female fetus undergo demethylation reprogramming for the next generation, isolated irom the vari ance of the outside world in a stabilized in utero environment, as does the male germ line after postetlization in the zygote (23) In addition to the early phases of methyia tion reprogramming that act on genomic imprinting and genes that are coadapted for developing the hypothalamus and. placenta (64), certain genes that are destined to form. ‘the neocortex have alate phase of reprog: ramming in the postnatal period (63). In deed, the major part of neocortical brain development occurs postnatally, and its fanctioning i designed to adapt to the social and physical enviconment. Unlike geem line cells, cortical neurons require a capacity to reprogram by demethylation/methylation throughout aduit life. Demethybtion (Teel) jccurs during the process of hippocampal learning and memory (66) and during adult neurogenesis, which impacts on the olfactory system (67). Learning and memory require ‘neuronal activity, which can strengten sy aptic connections and weaken other synaptic connection strengths through a process of synaptic plasticity. The signaling events trig gered by synaptic activity involve Ca2* entry to the neuron via a range of neural receptors (NMDA and GABA receptors featuring pre dominantly), leading to changes in neuro transmitter release and epigenetic changes to DNA methylation, acetylation, and hydroxy ‘methylation. Through these epigenetic changes, neurons gain high plasticity to integrate and store-new information. Inhibition of DNA methyltransferases (Dnmt) disrupts long-term potentiation in the hippocam- pus and alters methylation within the pro moter regions of Reelin and brain-derived PNAS | June2,2015 | vol 112 | no.22 | 6784 Eoh i” neurotrophic factor (BDNF), two hippo ccampal genes engaged with synaptic plas- ticity (66), Just as etl, -2, and -3 methyleytosine ioxygenases play an integral role in the reprogramming ofthe embryonic germ lin their function is crucial to neurons and the mechanisms underpinning learning and memory (68). Tet-mediated demethylation isfound at its highest levels in the brain and is thought to influence both passive deme- thylation by acting on the Dnmt enzymes and to serve as an intermediary in active demethylation. ‘etl has been shown to promote DNA demethylation in the adult brain at actvity-dependent synapses in the hippocampus (69). ‘Analysis of Tet! knockout mice demon- strate down-regulation of hippocampal genes ‘engaged with neural activity (Npast fos, and {An), accompanied by impaired memory ex tinction and abnormal longterm depression (70). Tet? mutant mice have also been shown to have impaired short term learning and memory (71), whereas animals overexpressing ‘Tett in the hippocampus have long-term ‘memory impairment (71), No abnormalities ‘were revealed in the developing brain, hip pocampal neurogenesis, or neuronal differ entiation of Tet knockout mice, suggesting level of redundancy across the methyl to sine dioxygenase ‘A number of genes that are destined to be expressed in the brain escape demethylation in the germ line 25). Such genes are mainly associated with repeat elements, but also in ‘ude neurally functioning genes. This find ing would suggest the possiblity for neural specific expression of genes that underake clemethylation ata later phase in the brain ‘This would meet the need for conserving methylation into the later stages of neocor- tical development when postmeiotic cal di Vision and demethylation coincide with up regulation ofthe base excision repair pathway (72). Such events would serve to protect the brain against cumulative genetic damage Certain region ofthe bran (hippocampus, ‘factory bul) continue neurogenesis in the adult brain. Overexpression of Tet has been shown to dscupt the successful anatomical Integration ofthe newly generated olfactory receptor neurons (67). ‘The continuous re newal ofthese sensory neurons is required to maintain neural plasticity in the olfactory system, the most important sensory system for the majority of mammals. Because con- tinual exposure of chemoreceptor neurons to ‘environmental toxins results in their death, Uheir regeneration and replacement through ‘ut lif is essential to continue chemosensory function. ‘This regeneration of the receptor 6782 | mum prascregid0 107Spnas 501482112 neurons also requires some reorganization of neurons in the circuitry atthe initial ray in the olfactory bulb. Tet3 action is therelore important to ensure the functional integra tion of these receptor neurons to sustain sensory recognition. Neural Development and Neurodevelopmental Disorders “The in utero development of the brain com- prises the formation of thousands of unique call types, each one with its own unique st fof fictional and melecula properties. This complex developmental event is orchestrated first by gastnlation (which specifies the neuroectoderm) and then by a set of over- lapping gradints of signaling molecules. OF these, the most prominent is sonic hedgehog secreted by the notochord, which species dlorsovental identity, but Wnt, BMP, and PGE signaling pathways all play major roles in the process as wall (73-8). In addition to the patterning ofthese gra dents, many neurons are specified in eyions fof the brain distant from where they will reside, and have to undergo @ complex mi- gation process, following local cues to ative to their destination (82). Double-Stranded RNA-Specific Endori- bonuclease (Droshay/DiGeorge Critical Region 8. Bocause of the sheer volume of events and cell types, the epigenetics of rental development are difficlt to catalog and elucidate, Tis task, however, is medi cally crucial, as deviations from normal developmental processes can lead to the personal anguish of a variety of neuro- developmental disorders, each of which can ‘ost, over a lifetime, milions of dollars per child. Some progress is being made, starting With the mechanisms that cause neuro- developmental defects of conditions such as Rett’s syndrome (83), which is epigenetic in origin, MeCP2, a methylated DNA binding protcin, is recognized as a transcriptional repressor, dysfunctions of which lead to the neurodevelopment disorders of Rett syn- ‘drome and autism spectrum disorder (84). Recently, MeCP2 was found to be involved in posttranscriptional gene regulation by repressing the processing of nuclear micto: RNAs through involvement of the Droshal Deer complex. MeCP2 binds directly to Dgcs8 (DiGeorge critical region 8), a key ‘component ofthe nuclear miRNA processing pathivay and interferes with the assembly of Drosha/Dgers (84). Interaction of DGCRS with MeCP2 (84, 85) is independent on the DNA binding domain of MeCP2, but controls microRNA. processing via the di- rect interaction with Dger8, One particular microRNA known tobe regulated by Dger8 is miR134, which regulates the expression ‘of Nanog and BDNF (86). Noncodling RNAS are gaining recognition in the context of brain development and psychiatric disorders, and are represented in this Sackler Caloquium by the paper of Goff etal. (87), Chromosomal 2241 1.2 deletion is the strongest known risk factor for schizo- pphrenia (88). A primary candidate gene is Dyer8, which encodes a component of the RNA microprocessor complex (Drosha/ Dag), sential for microRNA biogenesis Mirt85 in particular is the most notable down-regulated miRNA in both the prefontal cortex and hippocampus, brain areas that are focal to schizophrenia research (89). Hap- loinsuiiceney of Dger8 in a mouse model reveal smaptc SERCA2 [sareo (endo) plas mmc reticulum Ca?" ATP asl overexpression, a finding that paral the cleat levels of Ca** ATP.ase found in schizophrenia brains (90). ‘Autism, Autism spectrum disorders present 2 significant and urgent challenge. Now re- ‘presenting more than 1 in every 100 children, diagnoses of autism depend on abnormal social behaviors, compulsive repetitive move ments, and language disorders, Because ofthe ‘overshelming evidence that various forms of prenatal stress contibute to autism (91-93), epigenctic modifications provide an obvious set of possible mechanisms for the spectrum of autism ike disorders. In some children with autism, mutations inthe genes encading subunits of SWUSNE like complexes, ATP-dependent chromatin remodelers seem to provide an epigenetic basis forthe disease (94). Similarly, Noonan and colleagues have reported that mutations in CHD8 (chromodomain/helicase-DNA binding protein) are nearly always autism- causing (95). In addition, CHDS hap Toinsufficiency results in down-regulation of ‘multiple genes involved in early brain de- velopment, Mutations in many of these genes are associated with autism (96). BDNF, act- ing through its effecs on several miRNAS bby affecting the Lin28/ethal-7 (Let-7) axis during development (97), exemplifies the kind of local chemical influence whose dyste- _glaton could contribute o autism. These data ‘comprise the vanguard of a large number of potential investigations into. the epigenetic mechanisms by which early stress could contribute to the development of symptoms ‘of autism, but much work remains 1 be done In fac, epigenetic phenomena may pro- vide part ofthe “missing heritability” in au- tism genetics, Although MZ twins clearly Ihave higher rates of autism diagnoses than ‘dizygotic twins or siblings (98), pure genetics Kevere ataoh i” only accounts for part of that diference “There ix a Gscrepancy: actual heritability is greater than ene ‘Potent penne 9D patos Ne! avg or 407 sind Muto UC Gan, Mommy 2012) Thee eet cs et et wh Co fem anemone targa ane 00 rc Mh 3 091 Col opr ed in ‘detonator Na N04 97 10 au Mra DOH) rage aca ogre, Set a cagor busing mason gpm x cos ‘hun rayne sem as acre 2 ‘pete terol ra Seng mon Co Sencar 3 192 ce tery SC DON) Han enbyesstctt (Sod nae aep depute he sero n bens felt et es Uk 209 23 13 ego tary 2 Ching Moyo fag CO ‘iar entyone son hed eprae racte ese Icon ern pment ew Ct 258 404 rie! (919 ard ape ete om ‘ru asa putts abe on et on Seletoninocrt nd tamrphe wesc Sm (ie ed 2010-4 orinetedtesartn el anbe nv cron Fon haan cmon som Mate a) 2-138. {6 41 ea ZO gece of ran spine es ‘hen poe spc es Sn 8 3 11 fncog Mt 2019) ead ene mead Bam eee ‘a bo POU) ocean ate a ‘scent dr tren {9 5 0 ean gate tna S| (hdd Soparacge are ccd yeaah ‘Scnewermnenannsmts stay. Md 9) 120 sone. 0000 Ingato a mre eter on Cob Tao {24 ang tl DOT Rap aes mace ter ter fom ran rp sm er are a 7708 {22 ect old ease enn eat ened ‘Soa rind SB 423 uta Ca Haman nyse cle GAA 124 Msn Xs. OO Often tee ‘Rem este pst ee praia es 15 foto es, 201) Oar ences man 196 het ea | Pe CO eta ‘ered sal fom prey 3 pep {27 veep Gta canton eget tases orien nl ster ts haan einen an (iva aed cre atop {20 horn ora cb ns pst ‘Meester aed 129 ret GO” Pac pe mao erin ania sem hrarsome hence O10, {34 metry bunts des I GOI) mpd and ‘Ciudad user eg tame ace tno gene 81 138 se 9 ap as Care Soy ¥ OO) ‘ei suo as 208 1 torre (012 cope dunia Sz y20920. 34 a0 Sta Sere tb 4 a 210 he I deomram geese» yo OG nO a ‘outa taster CP? Melt oc 2 15 saccy ca coLamoeRve oer oip rode st ‘Sirarben Song rane eed 4 {96 wa srg Sep fs B20 Tims bras ps om mses rarah {7 Meoenstot el OD Bowe waa in (Rear cone nae esha SO 138 ocak a O10 Kepaoes anpon ‘eainona ges ekg mercer RA (Baocnsratyinn mata eeepc hat eo ron ad ne tn Gres 2) alot unge OWL rene integer ea ert ame AP ete el {44 Ua 3 O01) 1 ance Att ar {2 sort saeme 20) te ma eon Ssfhon oC wg goth nd oer ene ‘a a A al NS) Mere raion te Nn 02 sch en oe i ead PNAS | June2,2015 | vol 112 | no. 22 | 6795 =