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1.
Genome Med ; 12(1): 88, 2020 10 14.
Article in English | MEDLINE | ID: mdl-33054850

ABSTRACT

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.


Subject(s)
Autism Spectrum Disorder/etiology , DNA Methylation , Epigenome , Fetal Blood , Genes, X-Linked , Neurogenesis , Autism Spectrum Disorder/diagnosis , Autism Spectrum Disorder/genetics , Biomarkers , Brain/metabolism , Child, Preschool , Computational Biology/methods , Epigenesis, Genetic , Erythrocyte Count , Female , Gene Expression Regulation , Humans , Infant , Infant, Newborn , Machine Learning , Male , Organ Specificity/genetics , Prognosis
2.
J Genet Couns ; 28(2): 456-465, 2019 04.
Article in English | MEDLINE | ID: mdl-30964579

ABSTRACT

The practice of genetic counseling is going to be impacted by the public's expectation that goods, services, information, and experiences happen on demand, wherever and whenever people want them. Building from trends that are currently taking shape, this article looks just over a decade into the future-to 2030-to provide a description of how the field of genetics and genetic counseling will be changed, as well as advice for genetic counselors for how to prepare. We build from the prediction that a large portion of the general public will have access to their digitized whole genome sequence anytime, any place, on any device. We focus on five topics downstream of this prediction: public health, personal autonomy, polygenic scores (PGS), evolving clinical practices, and genetic privacy.


Subject(s)
Genetic Counseling/trends , Public Health/trends , Female , Genetic Counseling/ethics , Humans , Public Health/ethics
3.
Hum Mol Genet ; 28(16): 2659-2674, 2019 08 15.
Article in English | MEDLINE | ID: mdl-31009952

ABSTRACT

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.


Subject(s)
Autistic Disorder/etiology , Cytochrome P-450 CYP2E1/genetics , DNA Methylation , Insulin Receptor Substrate Proteins/genetics , Maternal Exposure , Placenta/metabolism , Prenatal Exposure Delayed Effects , Autism Spectrum Disorder/etiology , Autistic Disorder/metabolism , Biomarkers , Cadherins/metabolism , Case-Control Studies , Child , Disease Susceptibility , Epigenesis, Genetic , Female , Gene Expression Profiling , Genome-Wide Association Study , Humans , Male , Pregnancy , Risk , Signal Transduction , Wnt Proteins/metabolism
4.
Nat Commun ; 9(1): 1616, 2018 04 24.
Article in English | MEDLINE | ID: mdl-29691382

ABSTRACT

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.


Subject(s)
Brain/physiology , Cerebral Cortex/metabolism , Circadian Rhythm , Prader-Willi Syndrome/genetics , RNA, Small Nucleolar/genetics , RNA, Small Nucleolar/metabolism , Animals , Chromatin/genetics , Chromatin/metabolism , DNA Methylation , Female , Gene Deletion , Humans , Male , Mice , Prader-Willi Syndrome/metabolism
5.
Epigenetics ; 13(3): 318-330, 2018.
Article in English | MEDLINE | ID: mdl-29613827

ABSTRACT

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.


Subject(s)
DNA Methylation/genetics , Embryonic Development/genetics , Epigenomics , Hypothalamus/growth & development , Animals , CpG Islands/genetics , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental/genetics , Humans , Hypothalamus/metabolism , Male , MicroRNAs/genetics , Mother-Child Relations , Neuronal Plasticity/genetics , Rats , Sequence Analysis, DNA , Sequence Analysis, RNA , Stress, Psychological/genetics , Whole Genome Sequencing
6.
Food Funct ; 9(2): 1187-1198, 2018 Feb 21.
Article in English | MEDLINE | ID: mdl-29372223

ABSTRACT

In our previous work in mice, we have shown that chronic consumption of a Western diet (WD; 42% kcal fat, 0.2% total cholesterol and 34% sucrose) is correlated with impaired cognitive function. Cognitive decline has also been associated with alterations in DNA methylation. Additionally, although there have been many studies analyzing the effect of maternal consumption of a WD on DNA methylation in the offspring, few studies have analyzed how an individual's consumption of a WD can impact his/her DNA methylation. Since the frontal cortex is involved in the regulation of cognitive function and is often affected in cases of cognitive decline, this study aimed to examine how chronic consumption of a WD affects DNA methylation in the frontal cortex of mice. Eight-week-old male mice were fed either a control diet (CD) or a WD for 12 weeks, after which time alterations in DNA methylation were analyzed. Assessment of global DNA methylation in the frontal cortex using dot blot analysis revealed that there was a decrease in global DNA methylation in the WD-fed mice compared with the CD-fed mice. Bioinformatic analysis identified several networks and pathways containing genes displaying differential methylation, particularly those involved in metabolism, cell adhesion and cytoskeleton integrity, inflammation and neurological function. In conclusion, the results from this study suggest that consumption of a WD alters DNA methylation in the frontal cortex of mice and could provide one of the mechanisms by which consumption of a WD impairs cognitive function.


Subject(s)
DNA Methylation , Diet, Western/adverse effects , Frontal Lobe/metabolism , Animals , Cognition , Eating , Female , Male , Mice , Mice, Inbred C57BL
7.
Epigenetics ; 12(11): 982-990, 2017.
Article in English | MEDLINE | ID: mdl-28925810

ABSTRACT

The dysregulation of genes in neurodevelopmental disorders that lead to social and cognitive phenotypes is a complex, multilayered process involving both genetics and epigenetics. Parent-of-origin effects of deletion and duplication of the 15q11-q13 locus leading to Angelman, Prader-Willi, and Dup15q syndromes are due to imprinted genes, including UBE3A, which is maternally expressed exclusively in neurons. UBE3A encodes a ubiquitin E3 ligase protein with multiple downstream targets, including RING1B, which in turn monoubiquitinates histone variant H2A.Z. To understand the impact of neuronal UBE3A levels on epigenome-wide marks of DNA methylation, histone variant H2A.Z positioning, active H3K4me3 promoter marks, and gene expression, we took a multi-layered genomics approach. We performed an siRNA knockdown of UBE3A in two human neuroblastoma cell lines, including parental SH-SY5Y and the SH(15M) model of Dup15q. Genes differentially methylated across cells with differing UBE3A levels were enriched for functions in gene regulation, DNA binding, and brain morphology. Importantly, we found that altering UBE3A levels had a profound epigenetic effect on the methylation levels of up to half of known imprinted genes. Genes with differential H2A.Z peaks in SH(15M) compared to SH-SY5Y were enriched for ubiquitin and protease functions and associated with autism, hypoactivity, and energy expenditure. Together, these results support a genome-wide epigenetic consequence of altered UBE3A levels in neurons and suggest that UBE3A regulates an imprinted gene network involving DNA methylation patterning and H2A.Z deposition.


Subject(s)
Genomic Imprinting , Neurons/metabolism , Ubiquitin-Protein Ligases/genetics , Cell Line, Tumor , DNA Methylation , Histone Code , Humans , Ubiquitin-Protein Ligases/metabolism
9.
BMC Bioinformatics ; 18(1): 167, 2017 Mar 14.
Article in English | MEDLINE | ID: mdl-28288556

ABSTRACT

BACKGROUND: Guide Picker ( https://www.deskgen.com/guide-picker/ ) serves as a meta tool for designing CRISPR experiments by presenting ten different guide RNA scoring functions in one simple graphical interface. It allows investigators to simultaneously visualize and sort through every guide targeting the protein-coding regions of any mouse or human gene. RESULTS: Utilizing a multidimensional graphical display featuring two plots and four axes, Guide Picker can analyze all guides while filtering based on four different criteria at a time. Guide Picker further facilitates the CRISPR design process by using pre-computed scores for all guides, thereby offering rapid guide RNA generation and selection. CONCLUSIONS: The ease-of-use of Guide Picker complements CRISPR itself, matching a powerful and modular biological system with a flexible online web tool that can be used in a variety of genome editing experimental contexts.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats/genetics , RNA, Guide, Kinetoplastida/metabolism , User-Computer Interface , Animals , Gene Editing , Humans , Internet , Mice , RNA, Guide, Kinetoplastida/genetics
10.
Epigenetics ; 12(3): 206-214, 2017 03 04.
Article in English | MEDLINE | ID: mdl-28055307

ABSTRACT

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.


Subject(s)
DNA Methylation/genetics , Epidemiologic Studies , Epigenomics , High-Throughput Nucleotide Sequencing/methods , Algorithms , CpG Islands/genetics , Genome, Human , Genomics , Humans , Software
11.
Stem Cells ; 35(4): 981-988, 2017 04.
Article in English | MEDLINE | ID: mdl-28032673

ABSTRACT

Early embryonic stages of pluripotency are modeled for epigenomic studies primarily with human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSCs). For analysis of DNA methylation however, ESCs and iPSCs do not accurately reflect the DNA methylation levels found in preimplantation embryos. Whole genome bisulfite sequencing (WGBS) approaches have revealed the presence of large partially methylated domains (PMDs) covering 30%-40% of the genome in oocytes, preimplantation embryos, and placenta. In contrast, ESCs and iPSCs show abnormally high levels of DNA methylation compared to inner cell mass (ICM) or placenta. Here we show that dental pulp stem cells (DPSCs), derived from baby teeth and cultured in serum-containing media, have PMDs and mimic the ICM and placental methylome more closely than iPSCs and ESCs. By principal component analysis, DPSC methylation patterns were more similar to two other neural stem cell types of human derivation (EPI-NCSC and LUHMES) and placenta than were iPSCs, ESCs or other human cell lines (SH-SY5Y, B lymphoblast, IMR90). To test the suitability of DPSCs in modeling epigenetic differences associated with disease, we compared methylation patterns of DPSCs derived from children with chromosome 15q11.2-q13.3 maternal duplication (Dup15q) to controls. Differential methylation region (DMR) analyses revealed the expected Dup15q hypermethylation at the imprinting control region, as well as hypomethylation over SNORD116, and novel DMRs over 147 genes, including several autism candidate genes. Together these data suggest that DPSCs are a useful model for epigenomic and functional studies of human neurodevelopmental disorders. Stem Cells 2017;35:981-988.


Subject(s)
DNA Methylation/genetics , Dental Pulp/cytology , Genomic Imprinting , Stem Cells/cytology , Stem Cells/metabolism , Cell Line , Chromosome Duplication , Female , Genome, Human , Humans , Induced Pluripotent Stem Cells/metabolism , Placenta/metabolism , Pregnancy , Syndrome
12.
Cell Rep ; 17(11): 3035-3048, 2016 12 13.
Article in English | MEDLINE | ID: mdl-27974215

ABSTRACT

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.


Subject(s)
Autistic Disorder/genetics , Chromosome Duplication/genetics , DNA Methylation/drug effects , Epigenesis, Genetic , Autistic Disorder/pathology , Base Sequence/drug effects , Brain/drug effects , Brain/metabolism , Brain/pathology , Chromatin/drug effects , DNA Methylation/genetics , Gene Expression Regulation/drug effects , Gene-Environment Interaction , Genetic Association Studies , Genome, Human , Genomic Imprinting/genetics , Humans , Polychlorinated Biphenyls/toxicity , Polycomb Repressive Complex 1/genetics , Ubiquitin-Protein Ligases/genetics
13.
Hum Mol Genet ; 23(23): 6366-74, 2014 Dec 01.
Article in English | MEDLINE | ID: mdl-25008110

ABSTRACT

During postnatal development, neuronal activity controls the remodeling of initially imprecise neuronal connections through the regulation of gene expression. MeCP2 binds to methylated DNA and modulates gene expression during neuronal development and MECP2 mutation causes the autistic disorder Rett syndrome. To investigate a role for MeCP2 in neuronal circuit refinement and to identify activity-dependent MeCP2 transcription regulations, we leveraged the precise organization and accessibility of olfactory sensory axons to manipulation of neuronal activity through odorant exposure in vivo. We demonstrate that olfactory sensory axons failed to develop complete convergence when Mecp2 is deficient in olfactory sensory neurons (OSNs) in an otherwise wild-type animal. Furthermore, we demonstrate that expression of selected adhesion genes was elevated in Mecp2-deficient glomeruli, while acute odor stimulation in control mice resulted in significantly reduced MeCP2 binding to these gene loci, correlating with increased expression. Thus, MeCP2 is required for both circuitry refinement and activity-dependent transcriptional responses in OSNs.


Subject(s)
Methyl-CpG-Binding Protein 2/metabolism , Olfactory Bulb/metabolism , Sensory Receptor Cells/metabolism , Animals , Axons/metabolism , Axons/ultrastructure , Cadherins/metabolism , Methyl-CpG-Binding Protein 2/genetics , Mice , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Odorants , Olfactory Bulb/cytology , Protocadherins , Sensory Receptor Cells/ultrastructure , Transcription, Genetic
14.
Mol Autism ; 4(1): 3, 2013 Jan 25.
Article in English | MEDLINE | ID: mdl-23351786

ABSTRACT

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.

15.
Mol Cell Biol ; 32(14): 2894-903, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22615490

ABSTRACT

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.


Subject(s)
Methyl-CpG-Binding Protein 2/chemistry , Methyl-CpG-Binding Protein 2/metabolism , Amino Acid Sequence , Animals , Binding Sites/genetics , Brain/metabolism , Cell Differentiation , Cell Line , Cell Nucleus/metabolism , Ether-A-Go-Go Potassium Channels/genetics , Humans , Male , Methyl-CpG-Binding Protein 2/genetics , Mice , Molecular Sequence Data , Neurogenesis/genetics , Neurogenesis/physiology , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Phosphorylation , Promoter Regions, Genetic , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-ret/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Rett Syndrome/etiology , Rett Syndrome/genetics , Rett Syndrome/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic
16.
Hum Mol Genet ; 20(22): 4311-23, 2011 Nov 15.
Article in English | MEDLINE | ID: mdl-21840925

ABSTRACT

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.


Subject(s)
Autistic Disorder/metabolism , Brain/metabolism , Chromatin/metabolism , Chromosomes, Human, Pair 15/genetics , Receptors, Nicotinic/metabolism , Rett Syndrome/metabolism , Autistic Disorder/genetics , Binding Sites , Cell Line, Tumor , Chromatin/genetics , Humans , In Situ Hybridization, Fluorescence , Methyl-CpG-Binding Protein 2/genetics , Methyl-CpG-Binding Protein 2/metabolism , Prader-Willi Syndrome/genetics , Prader-Willi Syndrome/metabolism , Receptors, Nicotinic/genetics , Rett Syndrome/genetics , Reverse Transcriptase Polymerase Chain Reaction , alpha7 Nicotinic Acetylcholine Receptor
17.
J Hum Genet ; 56(7): 508-15, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21593744

ABSTRACT

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.


Subject(s)
DNA Copy Number Variations , Rett Syndrome/genetics , Blood Proteins/genetics , Chromatin Immunoprecipitation , Chromosomes, Human, Pair 1/genetics , Complement C3b Inactivator Proteins/genetics , Cytoskeletal Proteins/genetics , Female , Humans , Methyl-CpG-Binding Protein 2/genetics , Methyl-CpG-Binding Protein 2/metabolism , Phenotype
18.
Neurobiol Dis ; 43(1): 190-200, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21420494

ABSTRACT

Mutations in MECP2, encoding methyl CpG binding protein 2, cause the neurodevelopmental disorder Rett syndrome. MeCP2 is an abundant nuclear protein that binds to chromatin and modulates transcription in response to neuronal activity. Prior studies of MeCP2 function have focused on specific gene targets of MeCP2, but a more global role for MeCP2 in neuronal nuclear maturation has remained unexplored. MeCP2 levels increase during postnatal brain development, coinciding with dynamic changes in neuronal chromatin architecture, particularly detectable as changes in size, number, and location of nucleoli and perinucleolar heterochromatic chromocenters. To determine a potential role for MeCP2 in neuronal chromatin maturational changes, we measured nucleoli and chromocenters in developing wild-type and Mecp2-deficient mouse cortical sections, as well as mouse primary cortical neurons and a human neuronal cell line following induced maturation. Mecp2-deficient mouse neurons exhibited significant differences in nucleolar and chromocenter number and size, as more abundant, smaller nucleoli in brain and primary neurons compared to wild-type, consistent with delayed neuronal nuclear maturation in the absence of MeCP2. Primary neurons increased chromocenter size following depolarization in wild-type, but not Mecp2-deficient cultures. Wild-type MECP2e1 over-expression in human SH-SY5Y cells was sufficient to induce significantly larger nucleoli, but not a T158M mutation of the methyl-binding domain. These results suggest that, in addition to the established role of MeCP2 in transcriptional regulation of specific target genes, the global chromatin-binding function of MeCP2 is essential for activity-dependent global chromatin dynamics during postnatal neuronal maturation.


Subject(s)
Cell Differentiation/genetics , Cell Nucleolus/metabolism , Heterochromatin/metabolism , Methyl-CpG-Binding Protein 2/physiology , Neurons/metabolism , Animals , Animals, Newborn , Cell Line, Tumor , Cell Nucleolus/ultrastructure , Cells, Cultured , Female , Heterochromatin/genetics , Humans , Male , Methyl-CpG-Binding Protein 2/deficiency , Methyl-CpG-Binding Protein 2/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neurons/cytology
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