Placental DNA methylation levels at CYP2E1 and IRS2 are associated with child outcome in a prospective autism study.

DNA methylation acts at the interface of genetic and environmental factors relevant for autism spectrum disorder (ASD). Placenta, normally discarded at birth, is a potentially rich source of DNA methylation patterns predictive of ASD in the child. Here, we performed whole methylome analyses of placentas from a prospective study MARBLES (Markers of Autism Risk in Babies-Learning Early Signs) of high-risk pregnancies. A total of 400 differentially methylated regions (DMRs) discriminated placentas stored from children later diagnosed with ASD compared to typically developing controls. These ASD DMRs were significantly enriched at promoters, mapped to 596 genes functionally enriched in neuronal development, and overlapped genetic ASD risk. ASD DMRs at CYP2E1 and IRS2 reached genome-wide significance, replicated by pyrosequencing and correlated with expression differences in brain. Methylation at CYP2E1 associated with both ASD diagnosis and genotype within the DMR. In contrast, methylation at IRS2 was unaffected by within DMR genotype but modified by preconceptional maternal prenatal vitamin use. This study therefore identified two potentially useful early epigenetic markers for ASD in placenta.

15q11.2-13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain.

Copy number variations (CNVs) within human 15q11.2-13.3 show reduced penetrance and variable expressivity in a range of neurologic disorders. Therefore, characterizing 15q11.2-13.3 chromatin structure is important for understanding the regulation of this locus during normal neuronal development. Deletion of the Prader-Willi imprinting center (PWS-IC) within 15q11.2-13.3 disrupts long-range imprinted gene expression resulting in Prader-Willi syndrome. Previous results establish that MeCP2 binds to the PWS-IC and is required for optimal expression of distal GABRB3 and UBE3A. To examine the hypothesis that MeCP2 facilitates 15q11.2-13.3 transcription by linking the PWS-IC with distant elements, chromosome capture conformation on chip (4C) analysis was performed in human SH-SY5Y neuroblastoma cells. SH-SY5Y neurons had 2.84-fold fewer 15q11.2-13.3 PWS-IC chromatin interactions than undifferentiated SH-SY5Y neuroblasts, revealing developmental chromatin de-condensation of the locus. Out of 68 PWS-IC interactions with15q11.2-13.3 identified by 4C analysis and 62 15q11.2-13.3 MeCP2-binding sites identified by previous ChIP-chip studies, only five sites showed overlap. Remarkably, two of these overlapping PWS-IC- and MeCP2-bound sites mapped to sites flanking CHRNA7 (cholinergic receptor nicotinic alpha 7) encoding the cholinergic receptor, nicotinic, alpha 7. PWS-IC interaction with CHRNA7 in neurons was independently confirmed by fluorescent in situ hybridization analysis. Subsequent quantitative transcriptional analyses of frontal cortex from Rett syndrome and autism patients revealed significantly reduced CHRNA7 expression compared with controls. Together, these results suggest that transcription of CHRNA7 is modulated by chromatin interactions with the PWS-IC. Thus, loss of long-range chromatin interactions within 15q11.2-13.3 may contribute to multiple human neurodevelopmental disorders.

Investigation of modifier genes within copy number variations in Rett syndrome.

MECP2 mutations are responsible for two different phenotypes in females, classical Rett syndrome and the milder Zappella variant (Z-RTT). We investigated whether copy number variants (CNVs) may modulate the phenotype by comparison of array-CGH data from two discordant pairs of sisters and four additional discordant pairs of unrelated girls matched by mutation type. We also searched for potential MeCP2 targets within CNVs by chromatin immunopreceipitation microarray (ChIP-chip) analysis. We did not identify one major common gene/region, suggesting that modifiers may be complex and variable between cases. However, we detected CNVs correlating with disease severity that contain candidate modifiers. CROCC (1p36.13) is a potential MeCP2 target, in which a duplication in a Z-RTT and a deletion in a classic patient were observed. CROCC encodes a structural component of ciliary motility that is required for correct brain development. CFHR1 and CFHR3, on 1q31.3, may be involved in the regulation of complement during synapse elimination, and were found to be deleted in a Z-RTT but duplicated in two classic patients. The duplication of 10q11.22, present in two Z-RTT patients, includes GPRIN2, a regulator of neurite outgrowth and PPYR1, involved in energy homeostasis. Functional analyses are necessary to confirm candidates and to define targets for future therapies.

Cord blood DNA methylome in newborns later diagnosed with autism spectrum disorder reflects early dysregulation of neurodevelopmental and X-linked genes.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with complex heritability and higher prevalence in males. The neonatal epigenome has the potential to reflect past interactions between genetic and environmental factors during early development and influence future health outcomes. METHODS: We performed whole-genome bisulfite sequencing of 152 umbilical cord blood samples from the MARBLES and EARLI high-familial risk prospective cohorts to identify an epigenomic signature of ASD at birth. Samples were split into discovery and replication sets and stratified by sex, and their DNA methylation profiles were tested for differentially methylated regions (DMRs) between ASD and typically developing control cord blood samples. DMRs were mapped to genes and assessed for enrichment in gene function, tissue expression, chromosome location, and overlap with prior ASD studies. DMR coordinates were tested for enrichment in chromatin states and transcription factor binding motifs. Results were compared between discovery and replication sets and between males and females. RESULTS: We identified DMRs stratified by sex that discriminated ASD from control cord blood samples in discovery and replication sets. At a region level, 7 DMRs in males and 31 DMRs in females replicated across two independent groups of subjects, while 537 DMR genes in males and 1762 DMR genes in females replicated by gene association. These DMR genes were significantly enriched for brain and embryonic expression, X chromosome location, and identification in prior epigenetic studies of ASD in post-mortem brain. In males and females, autosomal ASD DMRs were significantly enriched for promoter and bivalent chromatin states across most cell types, while sex differences were observed for X-linked ASD DMRs. Lastly, these DMRs identified in cord blood were significantly enriched for binding sites of methyl-sensitive transcription factors relevant to fetal brain development. CONCLUSIONS: At birth, prior to the diagnosis of ASD, a distinct DNA methylation signature was detected in cord blood over regulatory regions and genes relevant to early fetal neurodevelopment. Differential cord methylation in ASD supports the developmental and sex-biased etiology of ASD and provides novel insights for early diagnosis and therapy.

Snord116-dependent diurnal rhythm of DNA methylation in mouse cortex.

Rhythmic oscillations of physiological processes depend on integrating the circadian clock and diurnal environment. DNA methylation is epigenetically responsive to daily rhythms, as a subset of CpG dinucleotides in brain exhibit diurnal rhythmic methylation. Here, we show a major genetic effect on rhythmic methylation in a mouse Snord116 deletion model of the imprinted disorder Prader-Willi syndrome (PWS). More than 23,000 diurnally rhythmic CpGs are identified in wild-type cortex, with nearly all lost or phase-shifted in PWS. Circadian dysregulation of a second imprinted Snord cluster at the Temple/Kagami-Ogata syndrome locus is observed at the level of methylation, transcription, and chromatin, providing mechanistic evidence of cross-talk. Genes identified by diurnal epigenetic changes in PWS mice overlapped rhythmic and PWS-specific genes in human brain and are enriched for PWS-relevant phenotypes and pathways. These results support the proposed evolutionary relationship between imprinting and sleep, and suggest possible chronotherapy in the treatment of PWS and related disorders.

Experience-dependent neuroplasticity of the developing hypothalamus: integrative epigenomic approaches.

Augmented maternal care during the first postnatal week promotes life-long stress resilience and improved memory compared with the outcome of routine rearing conditions. Recent evidence suggests that this programming commences with altered synaptic connectivity of stress sensitive hypothalamic neurons. However, the epigenomic basis of the long-lived consequences is not well understood. Here, we employed whole-genome bisulfite sequencing (WGBS), RNA-sequencing (RNA-seq), and a multiplex microRNA (miRNA) assay to examine the effects of augmented maternal care on DNA cytosine methylation, gene expression, and miRNA expression. A total of 9,439 differentially methylated regions (DMRs) associated with augmented maternal care were identified in male offspring hypothalamus, as well as a modest but significant decrease in global DNA methylation. Differentially methylated and expressed genes were enriched for functions in neurotransmission, neurodevelopment, protein synthesis, and oxidative phosphorylation, as well as known stress response genes. Twenty prioritized genes were identified as highly relevant to the stress resiliency phenotype. This combined unbiased approach enabled the discovery of novel genes and gene pathways that advance our understanding of the epigenomic mechanisms underlying the effects of maternal care on the developing brain.

A comparison of existing global DNA methylation assays to low-coverage whole-genome bisulfite sequencing for epidemiological studies.

DNA methylation is an epigenetic mark at the interface of genetic and environmental factors relevant to human disease. Quantitative assessments of global DNA methylation levels have therefore become important tools in epidemiology research, particularly for understanding effects of environmental exposures in complex diseases. Among the available methods of quantitative DNA methylation measurements, bisulfite sequencing is considered the gold standard, but whole-genome bisulfite sequencing (WGBS) has previously been considered too costly for epidemiology studies with high sample numbers. Pyrosequencing of repetitive sequences within bisulfite-treated DNA has been routinely used as a surrogate for global DNA methylation, but a comparison of pyrosequencing to WGBS for accuracy and reproducibility of methylation levels has not been performed. This study compared the global methylation levels measured from uniquely mappable (non-repetitive) WGBS sequences to pyrosequencing assays of several repeat sequences and repeat assay-matched WGBS data and determined uniquely mappable WGBS data to be the most reproducible and accurate measurement of global DNA methylation levels. We determined sources of variation in repetitive pyrosequencing assays to be PCR amplification bias, PCR primer selection bias in methylation levels of targeted sequences, and inherent variability in methylation levels of repeat sequences. Low-coverage, uniquely mappable WGBS showed the strongest correlation between replicates of all assays. By using multiplexing by indexed bar codes, the cost of WGBS can be lowered significantly to improve the accuracy of global DNA methylation assessments for human studies.

Cumulative Impact of Polychlorinated Biphenyl and Large Chromosomal Duplications on DNA Methylation, Chromatin, and Expression of Autism Candidate Genes.

Rare variants enriched for functions in chromatin regulation and neuronal synapses have been linked to autism. How chromatin and DNA methylation interact with environmental exposures at synaptic genes in autism etiologies is currently unclear. Using whole-genome bisulfite sequencing in brain tissue and a neuronal cell culture model carrying a 15q11.2-q13.3 maternal duplication, we find that significant global DNA hypomethylation is enriched over autism candidate genes and affects gene expression. The cumulative effect of multiple chromosomal duplications and exposure to the pervasive persistent organic pollutant PCB 95 altered methylation of more than 1,000 genes. Hypomethylated genes were enriched for H2A.Z, increased maternal UBE3A in Dup15q corresponded to reduced levels of RING1B, and bivalently modified H2A.Z was altered by PCB 95 and duplication. These results demonstrate the compounding effects of genetic and environmental insults on the neuronal methylome that converge upon dysregulation of chromatin and synaptic genes.

MeCP2 modulates gene expression pathways in astrocytes.

BACKGROUND: Mutations in MECP2 encoding methyl-CpG-binding protein 2 (MeCP2) cause the X-linked neurodevelopmental disorder Rett syndrome. Rett syndrome patients exhibit neurological symptoms that include irregular breathing, impaired mobility, stereotypic hand movements, and loss of speech. MeCP2 protein epigenetically modulates gene expression through genome-wide binding to methylated CpG dinucleotides. While neurons have the highest level of MeCP2 expression, astrocytes and other cell types also express detectable levels of MeCP2. Recent studies suggest that astrocytes likely control the progression of Rett syndrome. Thus, the object of these studies was to identify gene targets that are affected by loss of MeCP2 binding in astrocytes. METHODS: To identify gene targets of MeCP2 in astrocytes, combined approaches of expression microarray and chromatin immunoprecipitation of MeCP2 followed by sequencing (ChIP-seq) were compared between wild-type and MeCP2-deficient astrocytes. MeCP2 gene targets were compared with genes in the top 10% of MeCP2 binding levels in gene windows either within 2 kb upstream of the transcription start site, or the 'gene body' that extended from transcription start to end site, or 2 kb downstream of the transcription end site. RESULTS: A total of 118 gene transcripts surpassed the highly significant threshold (P < 0.005, fold change > 1.2) in expression microarray analysis from triplicate cultures. The top 10% of genes with the highest levels of MeCP2 binding were identified in two independent ChIP-seq experiments. Together this integrated, genome-wide screen for MeCP2 target genes provided an overlapping list of 19 high-confidence MeCP2-responsive gene transcripts in astrocytes. Validation of candidate target gene transcripts by RT-PCR revealed that expression of Apoc2, Cdon, Csrp and Nrep were consistently responsive to MeCP2 deficiency in astrocytes. CONCLUSIONS: The first MeCP2 ChIP-seq and gene expression microarray analysis in astrocytes reveals a set of potential MeCP2 target genes that may contribute to normal astrocyte signaling, cell division and neuronal support functions, the loss of which may contribute to the Rett syndrome phenotype.

Phosphorylation of distinct sites in MeCP2 modifies cofactor associations and the dynamics of transcriptional regulation.

Mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2) lead to disrupted neuronal function and can cause the neurodevelopmental disorder Rett syndrome. MeCP2 is a transcriptional regulator that binds to methylated DNA and is most abundant in neuronal nuclei. The mechanisms by which MeCP2 regulates gene expression remain ambiguous, as it has been reported to function as a transcriptional silencer or activator and to execute these activities through both gene-specific and genome-wide mechanisms. We hypothesized that posttranslational modifications of MeCP2 may be important for reconciling these apparently contradictory functions. Our results demonstrate that MeCP2 contains multiple posttranslational modifications, including phosphorylation, acetylation, and ubiquitylation. Phosphorylation of MeCP2 at S229 or S80 influenced selective in vivo interactions with the chromatin factors HP1 and SMC3 and the cofactors Sin3A and YB-1. pS229 MeCP2 was specifically enriched at the RET promoter, and phosphorylation of MeCP2 was necessary for differentiation-induced activation and repression of the MeCP2 target genes RET and EGR2. These results demonstrate that phosphorylation is one of several factors that are important for interpreting the complexities of MeCP2 transcriptional modulation.