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The Ras GTPase-activating protein SYNGAP1 plays a central role in synaptic plasticity, and de novo SYNGAP1 mutations are among the most frequent causes of autism and intellectual disability. How SYNGAP1 is regulated during development and how to treat SYNGAP1-associated haploinsufficiency remain challenging questions. Here, we characterize an alternative 3' splice site (A3SS) of SYNGAP1 that induces nonsense-mediated mRNA decay (A3SS-NMD) in mouse and human neural development. We demonstrate that PTBP1/2 directly bind to and promote SYNGAP1 A3SS inclusion. Genetic deletion of the Syngap1 A3SS in mice upregulates Syngap1 protein and alleviates the long-term potentiation and membrane excitability deficits caused by a Syngap1 knockout allele. We further report a splice-switching oligonucleotide (SSO) that converts SYNGAP1 unproductive isoform to the functional form in human iPSC-derived neurons. This study describes the regulation and function of SYNGAP1 A3SS-NMD, the genetic rescue of heterozygous Syngap1 knockout mice, and the development of an SSO to potentially alleviate SYNGAP1-associated haploinsufficiency.
Assuntos
Processamento Alternativo , Deficiência Intelectual , Humanos , Camundongos , Animais , Regulação para Cima , Processamento Alternativo/genética , Neurônios/metabolismo , Camundongos Knockout , Deficiência Intelectual/genética , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genéticaRESUMO
BACKGROUND: Asthma is the most common chronic disease in children, occurring at higher frequencies and with more severe disease in children with African ancestry. METHODS: We tested for association with haplotypes at the most replicated and significant childhood-onset asthma locus at 17q12-q21 and asthma in European American and African American children. Following this, we used whole-genome sequencing data from 1060 African American and 100 European American individuals to identify novel variants on a high-risk African American-specific haplotype. We characterized these variants in silico using gene expression and ATAC-seq data from airway epithelial cells, functional annotations from ENCODE, and promoter capture (pc)Hi-C maps in airway epithelial cells. Candidate causal variants were then assessed for correlation with asthma-associated phenotypes in African American children and adults. RESULTS: Our studies revealed nine novel African-specific common variants, enriched on a high-risk asthma haplotype, which regulated the expression of GSDMA in airway epithelial cells and were associated with features of severe asthma. Using ENCODE annotations, ATAC-seq, and pcHi-C, we narrowed the associations to two candidate causal variants that are associated with features of T2 low severe asthma. CONCLUSIONS: Previously unknown genetic variation at the 17q12-21 childhood-onset asthma locus contributes to asthma severity in individuals with African ancestries. We suggest that many other population-specific variants that have not been discovered in GWAS contribute to the genetic risk for asthma and other common diseases.
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Asma , Negro ou Afro-Americano , Negro ou Afro-Americano/genética , Alelos , Asma/genética , Asma/metabolismo , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Proteínas Citotóxicas Formadoras de PorosRESUMO
INTRODUCTION: Variants in the tau gene (MAPT) region are associated with breast cancer in women and Alzheimer's disease (AD) among persons lacking apolipoprotein E ε4 (ε4-). METHODS: To identify novel genes associated with tau-related pathology, we conducted two genome-wide association studies (GWAS) for AD, one among 10,340 ε4- women in the Alzheimer's Disease Genetics Consortium (ADGC) and another in 31 members (22 women) of a consanguineous Hutterite kindred. RESULTS: We identified novel associations of AD with MGMT variants in the ADGC (rs12775171, odds ratio [OR] = 1.4, P = 4.9 × 10-8 ) and Hutterite (rs12256016 and rs2803456, OR = 2.0, P = 1.9 × 10-14 ) datasets. Multi-omics analyses showed that the most significant and largest number of associations among the single nucleotide polymorphisms (SNPs), DNA-methylated CpGs, MGMT expression, and AD-related neuropathological traits were observed among women. Furthermore, promoter capture Hi-C analyses revealed long-range interactions of the MGMT promoter with MGMT SNPs and CpG sites. DISCUSSION: These findings suggest that epigenetically regulated MGMT expression is involved in AD pathogenesis, especially in women.
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Various human diseases and pregnancy-related disorders reflect endometrial dysfunction. However, rodent models do not share fundamental biological processes with the human endometrium, such as spontaneous decidualization, and no existing human cell cultures recapitulate the cyclic interactions between endometrial stromal and epithelial compartments necessary for decidualization and implantation. Here we report a protocol differentiating human pluripotent stem cells into endometrial stromal fibroblasts (PSC-ESFs) that are highly pure and able to decidualize. Coculture of PSC-ESFs with placenta-derived endometrial epithelial cells generated organoids used to examine stromal-epithelial interactions. Cocultures exhibited specific endometrial markers in the appropriate compartments, organization with cell polarity, and hormone responsiveness of both cell types. Furthermore, cocultures recapitulate a central feature of the human decidua by cyclically responding to hormone withdrawal followed by hormone retreatment. This advance enables mechanistic studies of the cyclic responses that characterize the human endometrium.
Assuntos
Técnicas de Cocultura/métodos , Decídua/metabolismo , Endométrio/metabolismo , Fibroblastos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células Estromais/metabolismo , Feminino , HumanosRESUMO
There is a life-long relationship between rhinovirus (RV) infection and the development and clinical manifestations of asthma. In this study we demonstrate that cultured primary bronchial epithelial cells from adults with asthma (n = 9) show different transcriptional and chromatin responses to RV infection compared to those without asthma (n = 9). Both the number and magnitude of transcriptional and chromatin responses to RV were muted in cells from asthma cases compared to controls. Pathway analysis of the transcriptionally responsive genes revealed enrichments of apoptotic pathways in controls but inflammatory pathways in asthma cases. Using promoter capture Hi-C we tethered regions of RV-responsive chromatin to RV-responsive genes and showed enrichment of these regions and genes at asthma GWAS loci. Taken together, our studies indicate a delayed or prolonged inflammatory state in cells from asthma cases and highlight genes that may contribute to genetic risk for asthma.
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Asma/metabolismo , Cromatina/metabolismo , Células Epiteliais/fisiologia , Mucosa Respiratória/citologia , Rhinovirus/fisiologia , Adulto , Asma/genética , Células Cultivadas , Humanos , Transcrição GênicaRESUMO
The epigenome provides a substrate through which environmental exposures can exert their effects on gene expression and disease risk, but the relative importance of epigenetic variation on human disease onset and progression is poorly characterized. Asthma is a heterogeneous disease of the airways, for which both onset and clinical course result from interactions between host genotype and environmental exposures, yet little is known about the molecular mechanisms for these interactions. We assessed genome-wide DNA methylation using the Infinium Human Methylation 450K Bead Chip and characterized the transcriptome by RNA sequencing in primary airway epithelial cells from 74 asthmatic and 41 nonasthmatic adults. Asthma status was based on doctor's diagnosis and current medication use. Genotyping was performed using various Illumina platforms. Our study revealed a regulatory locus on chromosome 17q12-21 associated with asthma risk and epigenetic signatures of specific asthma endotypes and molecular networks. Overall, these data support a central role for DNA methylation in lung cells, which promotes distinct molecular pathways of asthma pathogenesis and modulates the effects of genetic variation on disease risk and clinical heterogeneity.
Assuntos
Asma/genética , Metilação de DNA , Epigênese Genética , Células Epiteliais/citologia , Adulto , Estudos de Casos e Controles , Feminino , Expressão Gênica , Humanos , Pulmão/citologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , TranscriptomaRESUMO
Transcriptional mediators of cell stress pathways, including HIF1α, ATF4, and p53, are key to normal development and play critical roles in disease, including ischemia and cancer. Despite their importance, mechanisms by which pathways mediated by these transcription factors interact with one another are not fully understood. In addressing the controversial role of HIF1α in cardiomyocytes (CMs) during heart development, we discovered a mid-gestational requirement for HIF1α for proliferation of hypoxic CMs, involving metabolic switching and a complex interplay among HIF1α, ATF4, and p53. Loss of HIF1α resulted in activation of ATF4 and p53, the latter inhibiting CM proliferation. Bioinformatic and biochemical analyses revealed unexpected mechanisms by which HIF1α intersects with ATF4 and p53 pathways. Our results highlight previously undescribed roles of HIF1α and interactions among major cell stress pathways that could be targeted to enhance proliferation of CMs in ischemia and may have relevance to other diseases, including cancer.
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Fator 4 Ativador da Transcrição/metabolismo , Proliferação de Células , Embrião de Mamíferos/citologia , Feto/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Hipóxia/fisiopatologia , Miócitos Cardíacos/citologia , Proteína Supressora de Tumor p53/metabolismo , Fator 4 Ativador da Transcrição/genética , Animais , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Embrião de Mamíferos/metabolismo , Feminino , Feto/metabolismo , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
Organisms with targeted genomic modifications are efficiently produced by gene editing in embryos using CRISPR/Cas9 RNA-guided DNA endonuclease. Here, to facilitate germline editing in rats, we used CRISPR/Cas9 to catalyze targeted genomic mutations in rat spermatogonial stem cell cultures. CRISPR/Cas9-modified spermatogonia regenerated spermatogenesis and displayed long-term sperm-forming potential following transplantation into rat testes. Targeted germline mutations in Epsti1 and Erbb3 were vertically transmitted from recipients to exclusively generate "pure," non-mosaic mutant progeny. Epsti1 mutant rats were produced with or without genetic selection of donor spermatogonia. Monoclonal enrichment of Erbb3 null germlines unmasked recessive spermatogenesis defects in culture that were buffered in recipients, yielding mutant progeny isogenic at targeted alleles. Thus, spermatogonial gene editing with CRISPR/Cas9 provided a platform for generating targeted germline mutations in rats and for studying spermatogenesis.
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Sistemas CRISPR-Cas , Mutação em Linhagem Germinativa , Espermatogônias/metabolismo , Animais , Células Cultivadas , Feminino , Marcação de Genes/métodos , Masculino , Ratos , Ratos Sprague-Dawley , Receptor ErbB-3/genética , Espermatogônias/transplanteRESUMO
Sleep disorders are highly prevalent during late pregnancy and can impose adverse effects, such as preeclampsia and diabetes. However, the consequences of sleep fragmentation (SF) on offspring metabolism and epigenomic signatures are unclear. We report that physical activity during early life, but not later, reversed the increased body weight, altered glucose and lipid homeostasis, and increased visceral adipose tissue in offspring of mice subjected to gestational SF (SFo). The reversibility of this phenotype may reflect epigenetic mechanisms induced by SF during gestation. Accordingly, we found that the metabolic master switch Foxo1 was epigenetically misregulated in SFo livers in a temporally regulated fashion. Temporal Foxo1 analysis and its gluconeogenetic targets revealed that the epigenetic abnormalities of Foxo1 precede the metabolic syndrome phenotype. Importantly, regular physical activity early, but not later in life, reversed Foxo1 epigenetic misregulation and altered the metabolic phenotype in gestationally SF-exposed offspring. Thus, we have identified a restricted postnatal period during which lifestyle interventions may reverse the Foxo1 epigenetically mediated risk for metabolic dysfunction later in the life, as induced by gestational sleep disorders.
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Metabolismo Energético/genética , Epigênese Genética , Fatores de Transcrição Forkhead/genética , Fígado/metabolismo , Atividade Motora , Efeitos Tardios da Exposição Pré-Natal , Transtornos do Sono-Vigília/genética , Sono , Adiposidade , Fatores Etários , Animais , Biomarcadores/sangue , Glicemia/metabolismo , Feminino , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Idade Gestacional , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/fisiopatologia , Estilo de Vida , Lipídeos/sangue , Fígado/fisiopatologia , Camundongos Endogâmicos C57BL , Gravidez , Fatores de Risco , Comportamento de Redução do Risco , Transtornos do Sono-Vigília/sangue , Transtornos do Sono-Vigília/fisiopatologia , Aumento de PesoRESUMO
N(6)-methyladenosine is a prevalent internal modification in messenger RNA and non-coding RNA affecting various cellular pathways. Here we report the discovery of two additional modifications, N(6)-hydroxymethyladenosine (hm(6)A) and N(6)-formyladenosine (f(6)A), in mammalian messenger RNA. We show that Fe(II)- and α-ketoglutarate-dependent fat mass and obesity-associated (FTO) protein oxidize N(6)-methyladenosine to generate N(6)-hydroxymethyladenosine as an intermediate modification, and N(6)-formyladenosine as a further oxidized product. N(6)-hydroxymethyladenosine and N(6)-formyladenosine have half-life times of ~3 h in aqueous solution under physiological relevant conditions, and are present in isolated messenger RNA from human cells as well as mouse tissues. These previously unknown modifications derived from the prevalent N(6)-methyladenosine in messenger RNA, formed through oxidative RNA demethylation, may dynamically modulate RNA-protein interactions to affect gene expression regulation.
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Adenosina/análogos & derivados , Mamíferos/metabolismo , Proteínas/metabolismo , RNA/metabolismo , Adenosina/química , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Animais , Humanos , Cinética , Metilação , Camundongos , Modelos Biológicos , Simulação de Dinâmica Molecular , Oxirredução , Ligação Proteica , RNA/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Especificidade por SubstratoRESUMO
Genome-wide association studies (GWAS) routinely identify risk variants in noncoding DNA, as exemplified by reports of multiple single nucleotide polymorphisms (SNPs) associated with prostate cancer in five independent regions in a gene desert on 8q24. Two of these regions also have been associated with breast and colorectal cancer. These findings implicate functional variation within long-range cis-regulatory elements in disease etiology. We used an in vivo bacterial artificial chromosome (BAC) enhancer-trapping strategy in mice to scan a half-megabase of the 8q24 gene desert encompassing the prostate cancer-associated regions for long-range cis-regulatory elements. These BAC assays identified both prostate and mammary gland enhancer activities within the region. We demonstrate that the 8q24 cancer-associated variant rs6983267 lies within an in vivo prostate enhancer whose expression mimics that of the nearby MYC proto-oncogene. Additionally, we show that the cancer risk allele increases prostate enhancer activity in vivo relative to the non-risk allele. This allele-specific enhancer activity is detectable during early prostate development and throughout prostate maturation, raising the possibility that this SNP could assert its influence on prostate cancer risk before tumorigenesis occurs. Our study represents an efficient strategy to build experimentally on GWAS findings with an in vivo method for rapidly scanning large regions of noncoding DNA for functional cis-regulatory sequences harboring variation implicated in complex diseases.
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Cromossomos Humanos Par 8/genética , Elementos Facilitadores Genéticos/genética , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Cromossomos Artificiais Bacterianos , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Polimorfismo de Nucleotídeo Único , Proto-Oncogene MasRESUMO
The zinc finger transcription factor GLI1, which mediates Sonic hedgehog signaling during development, is expressed in several human cancers, including basal cell carcinoma, medulloblastoma, and sarcomas. We identified 147 genes whose levels of expression were significantly altered in RNA obtained from cells demonstrating a transformed phenotype with stable GLI1 expression or stable Ha-ras expression. Comparison of expression profiles from GLI1- and Ha-ras-expressing cells established a set of genes unique to GLI1-induced cell transformation. Thirty genes were altered by stable GLI1 expression, and 124 genes were changed by stable Ha-ras expression. Seven genes had altered expression levels in both GLI1- and Ha-ras-expressing cells. Genes whose expression was altered by GLI1 included cell cycle genes, cell adhesion genes, signal transduction genes, and genes regulating apoptosis. GLI1 consensus DNA-binding sequences were identified in the 5' regions of cyclin D2, IGFBP-6, osteopontin, and plakoglobin, suggesting that these genes represent immediate downstream targets. Gel shift analysis confirmed the ability of the GLI1 protein to bind these sequences. Up-regulation of cyclin D2 and down-regulation of plakoglobin were demonstrated in GLI1-amplified compared with non-amplified human rhabdomyosarcoma cells. Many of the GLI1 targets with known function identified in this study increase cell proliferation, indicating that GLI1-induced cell transformation occurs through multiple downstream pathways.