Epigenetic Mechanisms in Autism Spectrum

  

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• , Edwin H. Cook
• ,
• The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, Chicago, Illinois, USA _† Institute for Juvenile Research, Department of Psychiatry, University of Illinois at Chicago, Chicago, Illinois, USA
₁ Corresponding author: e-mail address: dgrayson@psych.uic.edu

  4. Epigenetic Dysregulation of ASD Candidate Genes 218

  References 231

  Acknowledgments 231

  6. Conclusions 229

  5. Environmental Model of Autism 227

  4.9 SH3 and multiple ankyrin repeat domains (SHANK3) 226

  4.8 Engrailed-2 (EN-2) 225

  4.7 Ubiquitin–protein ligase E3A (UBE3A) 224

  4.6 Brain-derived neutrophic factor (BDNF) 223

  4.5 Oxytocin receptor (OXTR) 223

  4.4 GABA β3 222

  4.3 Reelin 220

  4.2 GAD67 (GAD1) 219

  4.1 GABAergic genes 218

  3.3 Chromodomain helicase DNA-binding protein 8 217

  CHAPTER SIX

  3.2 DNA topoisomerase 216

  3.1 Methyl-CpG-binding protein 2 (MECP2) 214

  3. Genetic Defects with Epigenetic Implications 213

  2.3 DNA hydroxymethylation 212

  2.2 DNA methylation 209

  2.1 Histones 206

  2. Molecular Aspects of Epigenetic Mechanisms 206

  1. Introduction 204

  Contents

  Dennis R. Grayson

  Alessandro Guidotti

  Adrian Zhubi

  

Epigenetic Mechanisms in Autism

Spectrum Disorder

  Abstract Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by impaired social interactions, language deficits, as well as restrictive or repetitive behav- iors. ASD is clinically heterogeneous with a complex etiopathogenesis which may be

  Adrian Zhubi et al.

  conceptualized as a dynamic interplay between heterogeneous environmental cues and predisposing genetic factors involving complex epigenetic mechanisms. Inherited and de novo copy number variants provide novel information regarding genes contrib- uting to ASD. Epigenetic marks are stable, yet potentially reversible, chromatin modifi- cations that alter gene expression profiles by locally changing the degree of nucleosomal compaction, thereby opening or closing promoter access to the transcrip- tional machinery. Here, we review progress on studies designed to provide a better understanding of how epigenetic mechanisms impact transcriptional programs oper- ative in the brain that contribute to ASD.

1. INTRODUCTION

  Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by symptoms that include deficits in social interactions, under-developed communication skills, and restrictive or repetitive behav- iors. Population-wide prevalence of ASD is approximately 1% and it is more common in males than females (4:1)

  

). ASD is clinically and etiologically heteroge-

  neous with many of the diagnostic symptoms showing considerable variation in severity. Approximately 5–20% of cases involve large effect

  

de novo copy number variants. Genome-wide association studies are fre-

  quently used to compare the frequencies of single-nucleotide polymor- phisms (SNPs) in ASD DNA samples. While no replicable single SNP variants have been identified, the cumulative contributions of inherited genetic variation over many small effect loci has recently been estimated to be as high as 40%

  Microarrays are used extensively to map the large number of copy num- ber variants (CNVs) in the human population These mutations are variable in length and are either inherited or arise due to spontaneous (de novo) duplication, insertion, or deletion across the genome. Given the phenotypic overlap, it is not surprising that CNVs tend to be more frequent in patients with psychiatric disorders of neu- rodevelopmental origin such as intellectual disability/mental retardation (including patients with learning deficits), schizophrenia (SZ), and ASD ( Various tests of cognitive function show that control subjects known to be carrying CNVs that confer

  Epigenetic Mechanisms in Autism Spectrum Disorder

  intermediate between SZ and population controls ). Moreover, CNVs do not all affect the same cognitive domains and vary con- siderably from one mutation to another ( ). Two important goals of clinical geneticists are (1) to improve CNV detection and (2) to improve the phenotyping of CNV carriers that also exhibit psy- chotic symptoms. Recent analyses of topological networks derived from ASD CNVs and mouse functional genomics are being used to unveil highly detailed ASD-associated interaction networks that allow testing of novel hypotheses regarding cellular signaling and biological function

  One of the more frequently observed CNVs associated with high pen- etrance for ASD is the maternal duplication of chromosome 15q11-13. A second well-known CNV is a 600 kb microdeletion/microinsertion at chromosome 16p11.2 which occurs in some 1% of sporadic ASD cases ). Syndromic ASD, which occurs in 10% of cases, is observed when diagnostic behaviors are comorbid with a recognized syn- drome. Some of the monogenic (syndromic) conditions associated with the ASD phenotype are Rett (RTT) syndrome (MECP2 gene;

  

neurofibromatosis (NF1), Timothy syndrome TSC2;

  (CACNA1; ). Each of the above conditions exhibits phenotypes that overlap with ASD and hence offer important insight into how the corresponding genes contribute to pathogenesis.

  Several twin studies have reported concordance rates between MZ twins at between 70% and 90%, while DZ twin concordance rates vary from 6% to 10%, with a more than 20-fold increased risk for siblings (

  

Phenotypic dis-

  cordance between MZ twins is often associated with de novo mutations

  

) and numerous nonshared environmental factors including in utero

  growth restrictions (these include uneven blood supply, placental dysfunc- tion, differential allelic expression, etc., and stochastic noise (

  

In addition, numerous pre and

  perinatal factors, e.g., low birth weight and prematurity, are among multiple factors associated with higher risk of ASD

  

). These observations are consistent with the hypothesis that environ-

  mental factors contribute to ASD and support an epigenetic component in

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  allelic origin (imprinting) or the effects of DNA methylation/hydro- xymethylation on the epigenome. Moreover, epigenetic factors also mani- fest at multiple additional levels including histone tail modifications, variations in methyl DNA-binding proteins, etc. Recently, there has been a growth in interest in studies of the involvement of epigenetic mechanisms in the pathophysiology of ASD. The overlapping phenotypic features of ASD that are shared with related neurodevelopmental disorders may be explained through altered gene expression profiles filtered through various epigenetic mechanisms

  

In other words, as researchers begin to compare

  the gene expression networks between different neurodevelopmental disor- ders, differences in specific biological pathways and brain regions that reflect the phenotypes associated with each disease should become apparent (

  

In summary, one plausible mechanistic approach to understanding

  the etiology of ASD supports the concept that environmental/epigenetic perturbations incurred during early nervous system development operate on and enhance the contributions of a large number of susceptibility genes identified as either inherited or de novo mutations which themselves may influence epigenetic regulation (see ). Here, we summarize progress on epigenetic mechanisms operative in brain with the goal of understanding the distinct or overlapping features of these mechanisms in the etiopathogenesis of ASD. We provide a basic overview of histone modifi- cations, DNA methylation, and hydroxymethylation before proceeding onto subsequent topics. While we have not discussed the impact of micro- RNAs (miRNAs), long noncoding RNAs (ncRNAs), and enhancer RNAs on transcriptional regulation, there have been several recent reviews on these subjects (

  

).

2. MOLECULAR ASPECTS OF EPIGENETIC MECHANISMS

2.1. Histones

  Histones are small basic proteins composed of a globular core, C-terminal and N-terminal tails. DNA is a double-stranded helix wrapped around a his- tone octamer composed of two copies each of histones H2a, H2b, H3, and

  Epigenetic Mechanisms in Autism Spectrum Disorder

Figure 6.1 The interplay between environment, genetics, and epigenetics. The com- plex nature of ASD etiopathogenesis may be better appreciated when conceptualized

  as a dynamic interplay between the triad of genetic factors, epigenetic mechanisms, and external or environmental factors. These contributing components interact with each other, amplifying the effects of each individual factor. Prenatal exposure to a mul- titude of environmental factors such as valproic acid, thalidomide, and ethanol are teratogenic/mutagenic particularly when consumed at critical times during gestation (see for review). Other factors such as very low birth weight, prematurity, parental age, and maternal rubella infection (as well as immune status) operate at the level of the epigenome and impact brain development as discussed in the text. The contribution of environmental factors to the risk of developing ASD has been recently reviewed The list of genes shown is partial and only a few examples have been listed. is the fundamental unit of chromatin. Arrays of nucleosomes are organized along the chromosome in compact structures which facilitate extensive interactions between the chromatin and various nuclear processes, including DNA replication, transcription, repair, and recombination (

  

). Adjacent nucle-

osomes are connected by a stretch of linker DNA from 30 to 80 bp in length.

  Nucleosomal DNA is in equilibrium between wrapped and unwrapped

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  these states accompany changes in transcription. Histones make contact with the DNA at multiple points, although amino terminal histone tails protrude from the histone core. Nucleosomal structures support the DNA in a mobile and interactive environment that provides a scaffold for the reversible epigenetic markings that induce local structural transitions. These epigenetic marks support DNA inside the nucleus in clusters of open (euchromatin), closed/restricted chromatin (heterochromatin), and several intermediate states associated with transitioning between these two

  

). The various epigenetic marks allow tran-

  scription factors, RNA polymerase II (PolII), and ancillary proteins access to cis-acting regulatory sequences proximal (promoters) to transcriptional start sites (TSSs). In addition, they facilitate long-range interactions between protein-bound distal enhancer elements and transcription factor-associated proximal promoter domains through the formation of topological loops organized in three dimensions

   ).

  Epigenetic modifications of chromatin at the molecular level are covalent posttranscriptional modifications of the histone proteins which occur largely at histone H3 and histone H4 and include acetylation, meth- ylation, demethylation, phosphorylation, ubiquitination, and sumoylation (

  

While acetylation occurs at lysines (K), methylation occurs

  at both lysines (K) and arginines (R) of the protruding histone tails ( ). Acetylation of histone lysine residues by histone acetylases neutralizes the positive charge of lysine by adding an acetyl group, thereby introducing repulsive forces between the histone tails and DNA. The net result of these electrorepulsive charges is a local opening of the chro- matin which allows transcription factor access. Removal of acetyl groups on modified lysines is catalyzed by families of histone deacetylases (HDACs). In contrast, the effect (transcriptional activation or repression) of histone meth- ylation, by lysine or arginine methyltransferases, is contextual and depends on the residues modified (H3K4, H3K9, H3K36, H3K79, H4K20, H3R8, H4R3) and the number of methyl groups (mono vs. di vs. tri) added. There are multiple, different lysine- and arginine methyltransferases that add methyl groups to histones. This dynamic interplay between DNA and his- tones plays a fundamental role in modulating the degree of chromatin com- paction in the vicinity of promoter domains ( The GC

  Epigenetic Mechanisms in Autism Spectrum Disorder

  modifications and increases the predictability of transcription at the corresponding sites (

  

). For example, promoters with a high GC content accompanied by

  H3K27ac and H4K20me1 are associated with active transcription (

  

). In contrast, low GC content promoters correlate with tran-

  scription most often when accompanied by H3K4me1, H3K79me1, and H3K9me3 ). There is an enormous diversity and growing complexity in the numbers of enzymes that (1) cat- alyze histone modifications (epigenetic writers), (2) remove these groups (erasers), and (3) proteins that recognize these modifications (readers) to effect downstream responses

2.2. DNA methylation

  Methylation of DNA is an important epigenetic mechanism involved in numerous processes including X-chromosome inactivation, imprinting, and the suppression of foreign DNA such as transposable elements, proviruses, and other sequences . The role of methylation in regulating gene expression has been increasing in interest in recent years

  

. Methylation consists of the addition of a methyl group

  to the C5 position of the cytosine base, an enzymatic reaction catalyzed by members of a family of evolutionarily related DNA methyltransferases (DNMTs) that include DNMT1, DNMT3A, and DNMT3B. DNMTs mediate the methyl group transfer from the donor, S-adenosyl methi- onine, to cytosines in DNA producing 5-methylcytosine (5-mC) and

  

S-adenosylhomocysteine. Until the advent of whole-genome bisulfite

  sequencing, DNA methylation in mammals was believed to largely occur at CpG dinucleotides. The costs associated with assessing genome-wide changes in DNA methylation facilitated the use of genome-wide methyl-CpG arrays which provide comprehensive coverage of CpG sites in the genome most rel- evant to transcription. While methyl-CpG arrays have proven both affordable and popular, they provide only a limited assessment of the levels of non-CpG methylation which is also an abundant modification. Recent studies indicate that methylation of CpH sites (H ¼ A, C, or T) occurs more frequently than CpG methylation in adult human neurons, although it is sparse in nonneuronal cells of the brain In adult mouse dentate neurons, 75% of methylated sites are in CpGs, while 25% are in CpH dinucleotides Interestingly, methylated CpH sites have been shown to repress transcription in vitro and are recognized by

  Adrian Zhubi et al.

  CpG islands are features of vertebrate genomes that were initially thought to be associated with the 5 -flanking regions of housekeeping and many tissue-specific genes They are stretches of DNA that have a higher than normal GC content over some 300 bp or more and are found to be proximal to 60–70% of gene pro- moters. These contiguous CpG-rich stretches are asymmetrically distributed throughout the genome ) and are largely refractory to DNA methylation. Thus, CpG island-containing pro- moters and enhancers are generally undermethylated (hypomethylated) consistent with a negative role for methylation in regulating transcription ( Based on data from recent genome-wide studies, CpG islands themselves appear to function as promoters and the high GC content plays a regulatory role in nucleosome positioning or phasing around TSSs ). This concept is consistent with the large num- bers of CpG islands located proximal to promoters. It has also been demon- strated that CpG islands delineate the borders of open chromatin domains, hence allowing transcription factor and RNA Pol II access to these open regions of DNA ( ).

  Recently, the zinc finger-CXXC (ZF-CXXC) domain family of pro- teins was shown to specifically recognize CpGs at nonmethylated DNA and recruit chromatin-modifying activities to CpG island elements (

  

The classic ZF-CXXC domain-containing

  protein, CFP1, binds unmethylated CpGs within CpG islands and recruits the SETD1 H3K4 methyltransferase complex, facilitating an enrichment of

3 H3K4me at nearby histones ( Thompson, Fazzari, & Greally, 2010 ). In this

  way, the binding of CFP1, and other methyl-CpG readers, provides a mech- anism of directing histone modifications to specific regions of chromatin

  

3

  proximal to CpG islands. H3K4me is permissive for transcription (

  

). Several additional ZF-CXXC domain-containing proteins

  include DNMT1, histone methyltransferases (MLL-1 and -2, KDM2 fam- ily), methyl-binding domain protein (MBD1), and Tet methylcytosine dioxygenases (TETs 1 and 3) all of which bind to DNA at CpGs and act to modify DNA or histones.

  Traditionally, DNA methylation has been characterized as a highly stable, epigenetic mark that maintains cell type-specific changes of gene expression and negatively correlates with transcription (

  

Recent observations suggest that DNA methyla-

  tion is dynamic and that an active DNA demethylation pathway exists that

  Epigenetic Mechanisms in Autism Spectrum Disorder

  pathway involves hydroxylation of 5-mC to form 5-hydroxymethylcytosine (5-hmC) by members of the Tet methylcytosine dioxygenase enzyme family (TETs 1–3, see below). TET proteins also further oxidize 5- hmC forming 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC), which are stable epigenetic marks that accumulate in brain. 5-fC and 5- caC are specifically recognized by thymine deglycosylase (TDG) and are removed by base excision repair (BER) forming unmodified cytosine ). The resultant TpG/mCpG or 5hmUpG/mCpG mis- matches are excised by DNA glycosylases (i.e., the methyl-CpG-binding domain protein 4 [MBD4] or thymine deglycosylase)

  

Mechanisms that utilize TDG-

  mediated excision of 5-formylcytosine and 5-carboxycytosine have received the most experimental support ( The rap- idly inducible growth arrest and DNA-damage-inducible protein 45 family (GADD45), a small family of immediate early genes (

  

is

  thought to coordinate this process by selectively targeting specific 5-hmCs and recruiting deaminases and glycosylases to the corresponding genomic regions

  Recently, a mechanism for protecting actively expressed genes from DNA methylation was described that links transcription, DNMT1, and extra coding RNA molecules (ecRNA; ). ecRNAs are ncRNAs that are widely expressed across the genome. While these are distinct from mRNAs and miRNAs, researchers are only beginning to understand how they regulate gene expression (see

  

for recent review). In addition to ecRNA protection from

  DNA methylation, there is evidence that RNA–DNA hybrids called “R- loops” also protect CpG islands from DNA methylation (

   ).

  The CCAAT/enhancer-binding protein, alpha (CEBPA) is a transcrip- tion factor that contains a basic leucine zipper domain and recognizes the CCAAT motif in the promoters of target genes. This protein modulates the expression of genes involved in cell cycle regulation in numerous tissues. A functional ecRNA derived from the CEBPA locus was identified and shown to regulate methylation levels of the corresponding gene ). DNMT1 is a multifunctional protein that has mul- tiple domains that allow it to interact with numerous molecules, including DNA, RNA, and other proteins ( ). A combination of RNA

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  DNMT1 binds to the CEBPA ecRNA and prevents methylation of the gene. DNMT1 sequestration of the ecRNA activates transcription produc- ing CEBPA RNA ( ). Moreover, genome-wide expression and methylation profiling of DNMT1-bound RNAs demon- strated that this protein–ecRNA association occurs across a large number of genes supporting a novel mechanism for blocking DNA methylation.

2.3. DNA hydroxymethylation

  5-hmC was first detected in the genomes of the T-even (T2, T4, and T6) bacteriophages some 60 years ago ( ). More recently, 5-hmC was detected in the mammalian genomes of cerebellar Purkinje cells and granule cells, and amounts of this base are approximately 10-fold more prominent in neurons than in peripheral tissues or embryonic stem cells ( The TET-methylcytosine dioxygenase pro- tein family (gene symbol ¼ TET, TET methylcytosine dioxygenase) are mammalian homologs of the trypanosome proteins, JBP1 and JBP2, and were discovered when TET1 was observed fused to MLL in acute myeloid leukemia ( ). There has been growing interest in the study of this family of enzymes because of their ability to bind unmethylated CpGs and oxidize 5-mC to 5-hmC, the first step by which 5-mC in the genome is demethylated. Additional TET-catalyzed oxidation products are stable and accumulate in the genome, including 5-fC and 5-caC (

  

). Interestingly, these additional oxidation states appear to be

  dynamically regulated during development and are recognized by specific epigenetic readers suggesting that they encode additional epigenetic infor- mation The high-affinity 5-hmC-binding protein, ubiquitin-like, containing PHD and RING finger domains, 2 (UHRF2) binds 5-hmC with high affinity and directs the sequential oxidation catalyzed by the TET proteins At many sites, 5-mC is removed following hydroxylation and this is accom- panied by transcription of proximal genes However, 5-hmC is a stable epigenetic mark and accumulates at promoters, gene bodies, splice junctions, and distal regulatory elements (

  

  The levels of 5-hmC are high, while the levels of 5-mC are reciprocally low near enhancer proximal transcription factor-binding sites

  Epigenetic Mechanisms in Autism Spectrum Disorder

  may represent an important neuronal- or cellular-specific epigenetic code That is, the accumulation of 5-hmC is likely a crit- ical step in the activation of cell-specific enhancers during differentiation or lineage specification

  As noted above, proteins that contain CXXC domains bind to unmethylated CpGs and recruit ancillary chromatin modifiers. It has been known for some time that proteins containing methyl DNA-binding domains (MBDs) as part of their structure, bind to methylated CpGs. These include MBD2 and MECP2. A recent proteomics analysis shows that MECP2 binds to both 5-mC and 5-hmC, while MBD3 binds primarily to 5-hmC ( ). Until recently, MECP2 was considered strictly as a global epigenetic repressor of gene expression. However, there is now a growing body of evidence supporting the idea that the function of

  

MECP2 is to modulate neuronal transcription in both directions. MECP2

  has a substantial role in modifying chromatin structure by binding to 5-hmC-enriched DNA regions within various gene domains, particularly in gene bodies where it correlates with those in the highest expression per- centiles The increase of 5-hmC and the concomitant depletion of 5-mC from gene body regions produces a high 5-hmC/5-mC ratio which shows the highest correlation with active gene expression in the major cell types of the cerebellum (Purkinje cells, granule cells, and Bergman glia; Hence, recent data suggest that the phenotypic variability of RTT and other ASD- related conditions may be related to alterations in the 5-hmC/5-mC ratio and differential binding of MECP2 which could result from mutations or posttranslational modifications in a cell-, region-, or circuit-specific manner.

3. GENETIC DEFECTS WITH EPIGENETIC IMPLICATIONS

  As noted in , phenotypic overlap with autism has been observed in several monogenic neurodevelopmental disorders, including FXS, RTT syndrome, MECP2 duplication syndrome (MDS), Down syn- drome, Angelman syndrome (AS), Prader–Willi syndrome, Williams syn- drome, fetal anticonvulsant syndrome, etc. Despite differences in the etiology of these conditions, the overlapping features evident on clinical pre- sentation strongly suggest the possibility that common neurobiological path- ways may be involved. In this context, associations between (1) RTT and

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3.1. Methyl-CpG-binding protein 2 (MECP2)

  More than 95% of RTT cases are caused by a mutation in the MECP2 gene RTT is an X-linked neurodevelopmental disorder that predominantly affects females and is characterized by autistic-like features, seizures, gait, ataxia, and stereotypical hand movements. MECP2 is a member of the MBD family of proteins and is an essential epigenetic regulator of human brain develop- ment. MECP2 functions in coordination with multiple chromatin transcrip- tional repressors and is linked to the Sin3A repressor and HDACs

  

In the

  human neuronal SH-SY5Y cell line, the 63% of promoter-bound MECP2 is associated with actively transcribed genes Chromatin immunoprecipitation assays of Ntera2 (NT2) cells show that MECP2, DNMT1, DNMT3a, and HDAC2 bind to the same stretch of DNA in the glutamic acid decarboxylase 67 (GAD1) and Reelin (RELN) promoters ( Studies of MECP2 knock-out and over-expressing mice show that MECP2 positively regulates the expression of a wide range of genes by associating with the transcrip- tional activator CREB1 and binding to cAMP response element-binding (CREB) sites ), (2) global alterations of chromatin condensation (4) pro- moting gene imprinting (

  

). Collectively, the evidence supports

  the concept that MeCP2 represents a complex and pleiotropic regulatory system that associates with 5-mC and 5-hmC and multiple ancillary proteins that serve to mediate downstream decisions regarding gene expression.

  MECP2 protein levels are highest in the central nervous system and increase postnatally during neuronal maturation

  

Optimal MECP2 levels are required for brain maturation during spe-

  cific developmental periods. Animal studies show that loss of MECP2 during postnatal stages causes the retraction of mature pyramidal neuron dendritic arbors, reductions in dendritic spine density, increases in neuronal cell den- sity, and a reduction in the levels of several synaptic proteins

  

Neuron- or region-specific MECP2 depletion reproduces some of

  Epigenetic Mechanisms in Autism Spectrum Disorder

  produces motor incoordination, while deletion in serotonergic neurons causes increased aggression Loss of MECP2 in the amygdala impairs amygdala-dependent learning and memory

  

while loss in the hypothalamus

  affects feeding behaviors, aggression, and stress responses (

  

). Moreover, deletion of MECP2 in GABAergic neurons supports

  an important role for the corresponding protein in the function of inhibitory neurons This study demonstrates that cortical wild-type GABAergic neurons express 50% more Mecp2 than non-GABAergic neurons. Mice with either a global loss of Mecp2 in GABAergic neurons or a conditional loss in a subset of forebrain structures (striatum and cortex) develop autistic-like features including stereotyped behaviors, deficits in social behaviors, motor function, learning and memory, and sensorimotor gating

  The role of genetic and epigenetic factors was recently examined in a MZ twin pair discordant for RTT that shared the same de novo mutation in exon 4 of MeCP2 Potential sources of discordance in RTT, in addition to non-shared environmental factors, include differen- tial patterns of X-chromosome inactivation. The exon 4 mutation in

  

MECP2 in these twins was paternal in origin and occurred during spermato-

  genesis. Differential DNA methylation patterns located upstream of several genes relevant to brain function and skeletal tissue (including Brain-type cre-

  

atine kinase (CKB), Fyn proto-oncogene, and Mohawk homeobox) were detected

  in skin fibroblasts. The corresponding mRNA levels also inversely correlate with DNA methylation levels CKB encodes a brain- selective isoform of creatine kinase which is important in brain energy homeostasis and to the function of GABAergic inter- neurons ( No additional DNA dif- ferences, aside from methylation, were identified between these twins in SNPs, insertion–deletion polymorphisms (indels), CNVs, or patterns of X-chromosome inactivation that might account for the above findings.

  Autistic-like features are characteristic of patients with MDS, including stereotypic hand movements, impaired speech development, or loss of speech after 4 years of age ( ). MDS patients also show stunted motor development, susceptibility to recur- rent respiratory infections, and anxiety (

  

While neurons isolated from MECP2-deficient mice show

  decreased excitation and increased inhibitory neurotransmission, MECP2

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  indicates that MECP2 gene dose bidirectionally affects excitatory transmis- sion ( ). In conclusion, monogenic defects in MECP2 expres- sion due to loss-of-function mutations (both SNPs and CNVs) present in RTT cases and gain-of-function mutations (MECP2 duplication and trip- lication syndrome) are likely relevant to the altered epigenetic signature observed in ASDs.

3.2. DNA topoisomerase

  Recent discoveries in chromatin biology have shown that the topology of DNA is complex and that local winding and unwinding of the DNA needs to occur to relax the torsional stress which accompanies nucleosome repositioning during transcription, as well as during DNA replication DNA topoisomerases 1 and 2 (TOP1 and TOP2) are vital to gene expression as they resolve the DNA supercoiling generated dur- ing transcription

  

). Topoisomerases interact

  directly with RNA PolII and enable transcription elongation over long stretches of DNA

  

topoisomerase

  was shown to facilitate the expression of long genes, >200 kb, many of which are associated with synaptic function and ASD. An impressive per- centage of long genes (27%) are also known ASD risk genes. By pharmaco- logically inhibiting TOP1 or knocking-down the expression of TOP1 or

  

TOP2b in neurons, the expression of long primary transcripts in mouse

  and human neurons are reduced The TOP1 inhibitor, topotecan, mediates transcriptional silencing of the UBE3A antisense tran- script (UBE3A-ATS) which is required for silencing the paternal allele of

  

Ube3a in mouse cortical neurons ). Independently,

  topotecan was shown to stabilize the formation of RNA:DNA hybrids (R-loops) at G-skewed repeat elements within paternal Snord116, a small nuclear ncRNA molecule inhibiting expression of Ube3a-ATS

  

). Topotecan has a similar effect on transcription of the human

UBE3A locus ( In the imprinting disorder AS, in which

UBE3A deficiency is mediated by mutations in the maternal allele,

  topotecan appears to be useful in activating expression of the paternal

  

UBE3A allele. That is, by inhibiting the expression of the antisense transcript

  (UBE3A-ATS), topoisomerase inhibitors might prove useful as a pharmaco- logical intervention to increase UBE3A mRNA levels.

  Epigenetic Mechanisms in Autism Spectrum Disorder

3.3. Chromodomain helicase DNA-binding protein 8

  The chromodomain helicase DNA-binding proteins are ATP-dependent chromatin-remodeling proteins that regulate transcription by alter the posi- tioning of nucleosomes over the DNA. Chromatin regulators contribute to both dynamic changes in gene expression and heritable states of gene expres- sion as required during brain development ( ). Chromodomain helicase DNA-binding protein 8 (CHD8) binds β-catenin (CTNNB1) and negatively regulates WNT signaling which plays a critical role in early vertebrate development and morphogenesis. Mice lacking Chd8 exhibit early embryonic death thought to the result of widespread p-53-induced apoptosis It was recently shown, by ChIP-on-chip analysis, that CHD8 binds to upward of 2000 transcrip- tionally active promoters in transformed cell lines

  

). Interestingly, the targets of CHD8 binding are also targets of

  the E2F family of transcription factors that regulate genes related to the cell cycle and proliferation ). The same study showed that CHD8 binds to promoters that are enriched in histone marks (H3K4me2 and H3K4me3) positively associated with transcription. Based on these and other studies, it appears that CHD8 acts as a negative transcrip- tional regulator in repressing p53 and CTNNB1 gene expression while positively modulating expression of large numbers of genes containing E2F-binding sites. Similar to MECP2 and TOP-1 and -2, CHD8 is involved in regulating large numbers of genes in different families at different stages during brain development.

  Numerous de novo mutations in CHD8 have been identified in ASD and enlarged head circumference (macrocephaly) was noted as a comorbid phe- notype

  

). CHD8 has also recently been shown to interact with

  a related chromodomain helicase DNA-binding protein 7 (CHD7) (

  

). CHD7 is deficient in approximately three-fourths of patients

  diagnosed with CHARGE syndrome, an autosomal dominant malformation syndrome (OMIM 214800). The disorder is characterized by variable com- binations of coloboma, heart defects, atresia of the choanae, retarded growth and development, genital hypoplasia, and ear anomalies and deafness. A yeast two-hybrid library screen using CHD8 identified CHD7 as an interacting partner which was confirmed by coimmunoprecipitation (

  

). It seems plausible that the complex phenotype observed in

  CHARGE could be the consequence of aberrant gene expression arising

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  from alterations in local chromatin conformation induced by these and other chromatin modifiers.

  Additional large-scale resequencing studies identified several genes, in addition to CHD8, including cadherin-associated protein, β1 (CTNNB1), and dual specificity tyrosine-phosphorylation-regulated kinase 1A in multiple patients (reviewed in

  

Protein-interacting networks built from mutations associated with

  ASD and intellectual disabilities indicate the presence of three large interconnected networks which include genes whose protein products function in (1) chromatin modification (primary hub gene ¼ CHD8), (2) WNT/CTNNB1 signaling pathway (primary hub ¼ CTNNB1), and (3) synaptic function (multiple hubs) ). As noted above, the downstream targets of the CHD proteins are only now being identified ( As more and more de novo mutations in ASD and related disorders are identified, genes that are epigenetically coupled to large downstream gene networks, such as CHD8 (and MECP2, TOP1, and

  

2), are likely to be identified and replicated in additional studies. Moreover,

  deciphering the function of the CHD proteins during brain development is still largely under explored.

  4. EPIGENETIC DYSREGULATION OF ASD CANDIDATE GENES

4.1. GABAergic genes

  A role for the GABAergic inhibitory system in the pathophysiology of ASD has been consistently reported based on the results of several postmortem human brain studies. These studies include reports of (1) reduction in the number of GABAergic Purkinje cells in cerebellar cortex (

  

  (2) reduction by 50% of the GABA-synthesizing enzymes glutamic acid decarboxylase 65 (GAD2) in cerebellum and GAD1 in parietal cortex of ASD patients ( ), (3) 40% down- regulation of GAD1 mRNA levels in cerebellar GABAergic Purkinje cells of ASD patients (4) reduction of GABA receptor binding in the hippocampus and anterior and posterior

  A

  cingulate cortices (5) reduction of GABRB3 receptor subunit expression in the cingulate cor- tex and cerebellar vermis of ASD (6) reduction in

  Epigenetic Mechanisms in Autism Spectrum Disorder

  (BA-40), (7) reduction of GABRA1 and GABRB3 in cerebellum, and (8) reduction of GABRA1 receptor subunit in the superior frontal cortices (BA-9) of ASD subjects ( It has been hypothesized that the dysfunction of GABA receptor subunits is

  A

  most likely responsible for the observed inhibitory signaling deficits which could explain the high comorbidity of RTT and ASD with seizures (80% in the RTT and 10–25% in ASD). In addition, 15–30% of children with epilepsy have ASD (

4.2. GAD67 (GAD1)

  Glutamate decarboxylase is the enzyme that catalyzes the decarboxylation of glutamate to form GABA. GAD67 and GAD65 were named based on the molecular size of the corresponding protein on Western blots

  

and this nomenclature has been replaced with

  the gene symbols GAD1 and GAD2, respectively. GAD67 and 65 are the protein products of different genes located on separate chromosomes and have distinct intracellular locations and distinct cofactor/substrate requirements ( ). Studies from our laboratories show that the GAD1 promoter is GC rich ) and its regulation negatively correlates with promoter proximal hypermethylation in SZ and bipolar (BP) subjects

  

GAD1 expression in ASD is also

  likely regulated by epigenetic mechanisms including DNMT-mediated DNA hypermethylation and DNA demethylation involving the initial hydroxylation of 5-mC to form 5-hmC by members of the TET protein family Both 5-hmC and TET1 are highly expressed in human cerebellum. Moreover, several studies have reported that Purkinje cell loss is one of the more consistent neuropatholog- ical findings in the postmortem cerebellum of ASD subjects

  

  To investigate the role of epigenetic mechanisms in regulating GAD1 dysfunction, we conducted experiments using postmortem cerebellar tissue obtained from Harvard Brain Tissue Resource Center, McLean Hospital, with approval of Autism Tissue Program ( ). The goal was to explore variations in 5-hmC and 5-mC in the regulatory regions of GAD1 and GAD2 whose expression is consistently reduced in postmor-

  Adrian Zhubi et al.

  TET1 mRNAs and also the binding of MECP2, DNMT1, and TET1 to pro- moter and gene body regions of GAD1 and 2 were examined. The results show an upregulation of TET1 mRNA and increased TET1 binding to the GAD1 promoter in ASD versus CON. The increased TET1 is associated with an enrichment of 5-hmC at the GAD1 promoter region ( In contrast, the levels of 5-mC at the promoter and gene body of GAD1 were unchanged. There were no significant differences in the levels of DNMT1 mRNA and protein binding to any GAD1 promoter regions. The increased levels of 5-hmC at the promoter in ASD may facilitate the binding of MECP2. This could reflect an increased binding affinity of a posttranslationally modified form of MECP2, since the protein levels are not different in ASD versus CON . In ASD, MECP2 likely acts to repress

  

GAD1 as MECP2 binding negatively correlates with the levels of GAD1

  mRNA. This repression shows some selectivity, since the levels of mRNA, MECP2 binding, and 5-hmC enrichment at the GAD2 promoter do not change in cerebellum of ASD versus CON subjects

4.3. Reelin

  RELN is a large extracellular matrix glycoprotein expressed in corticolimbic GABAergic neurons (

  

RELN that plays a pivotal role in

  neuronal migration and cortical lamination during embryonic development (

  

). Abnormal RELN levels associated

  with decreased dendritic spine densities have been identified in several psy- chiatric conditions including ASD, SZ, BP disorder, Alzheimer disease, etc. Several studies using postmortem tissue from ASD subjects have reported significant reductions in RELN protein (180-kDa protein fragment) in cerebella of subjects with ASD ( ), and a reduc- tion in RELN protein levels (410-, 310-, and 180-kDa protein fragments) in superior frontal cortex, parietal cortex, and cerebella of ASD subjects ( Similar studies have shown a significant reduction in

  

RELN mRNA levels in frontal cortex and cerebellum ASD subjects

  ( ). One mechanism to explain reduced expression of RELN mRNA in ASD is possibly RELN promoter

  Epigenetic Mechanisms in Autism Spectrum Disorder

Figure 6.2 Epigenetic changes in ASD brain. As indicated, the slide depicts mechanisms described in the text regarding the downregulation of gene expression in postmortem

  brain of ASD subjects. The drawing depicts the TET1-mediated hydroxymethylation and binding of MECP2 to hydroxymethylated and likely methylated DNA proximal to the GAD1 and RELN promoters. For a description of the data and methodology, see .

  Adrian Zhubi et al.

  (

  

Moreover, recent studies indicate that sex hormones

  might play a role in the methylation of the RELN promoter, particularly in the pubertal and postpubertal periods, which coincide with a worsening of autistic behaviors and the onset of SZ ( ). In our study of postmortem cerebellar samples, we observed that decreased RELN mRNA level in the cerebellum of ASD was associated with increased bind- ing of MECP2, which coincides with an enrichment of 5-hmC at the RELN promoter. This methylcytosine modification, catalyzed by TET1, is upregulated in ASD postmortem brain As shown in

  

increased 5-hmC at the RELN and GAD1 promoters is coincident

  with decreased transcription and decreased levels of the corresponding pro- teins. It is currently unclear why the increase in 5-hmC correlates with reduced expression in psychiatric disorders.

4.4. GABA β3

  15q11.2-13.3 is a genomic region prone to chromosomal rearrangements of various sizes that contains three distinct ASD susceptibility and CNV loci that vary in their genomic boundaries ( This is a complex locus that includes several imprinted genes, including

  

GABRB3, GABRA5, and GABRG3. Chromatin immunoprecipitation

  studies show that MECP2 binds to methylated sites in the first intron of

  The expres- GABRB3

  sion and binding of the sequence-specific transcription factor Specificity Pro- tein 1 (Sp1) in various brain regions of postmortem ASD subjects showed increased binding to DNA that negatively correlated with the levels of expression of several ASD candidate genes including GABRB3 and RELN ( ). Sp1 is a zinc finger transcription factor that binds to promoter GC-box elements and normally positively modulates gene expres- sion. Sp1 binding to promoters is influenced by environmental factors such as hypoxia, viral infection, glucose, and calcium levels. Results from these stud- ies show increased levels of Sp1 in the anterior cingulate cortex, but not in the motor cortex or thalamus of ASD subjects As a consequence of increased levels of Sp1, there is decreased GABRB3 expres- sion in motor cortex and decreased RELN mRNA in the anterior cingulate cortex of ASD subjects. Sp1, like MECP2, may represent a dynamic switch which affects the expression of multiple genes which participate in diverse