Transcriptomic analysis of mutant MECP2 and NEAT1 human neural cells
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PROVIDER: PRJEB75393 | ENA |
REPOSITORIES: ENA
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Project description:Rett Syndrome (RTT) is a severe neurological disorder predominantly affecting females, caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. Understanding the pathophysiology of RTT at a cellular and molecular level is crucial for the development of targeted therapies. This project aims to dissect the molecular underpinnings of RTT using a novel in vitro model system based on a commercially available human neural progenitor cell line, ReNCell. We have engineered multiple distinct ReNCell lines to mimic specific genetic alterations associated with RTT, providing a robust platform for mechanistic studies and drug screening. One cell line is a complete knockout of MECP2, serving as a model to investigate the effects of total loss of MeCP2 function. This model helps in understanding the full spectrum of MeCP2's roles in neural development and maintenance, and in identifying compensatory mechanisms that could be targeted therapeutically. The other line involves the knockdown of NEAT1, a long non-coding RNA known to be involved in the pathogenesis of several neurological disorders, including RTT. Recent studies suggest NEAT1 plays a critical role in the neuronal cellular response to MECP2 dysfunction. By reducing NEAT1 expression, we aim to elucidate its contribution to RTT pathology and explore its potential as a therapeutic target. Here we characterize the transcriptome of these cell lines, including the wild type (control), at the progenitor state and after 7 days of differentiation with three replicates each.
2024-11-17 | E-MTAB-14048 | biostudies-arrayexpress
Project description:Transcriptomic analysis of mutant MECP2 human neural progenitor cells
| PRJEB75392 | ENA
Project description:Rett Syndrome (RTT) is a severe neurological disorder predominantly affecting females, caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. Understanding the pathophysiology of RTT at a cellular and molecular level is crucial for the development of targeted therapies. Our project aims to dissect the molecular underpinnings of RTT using a novel in vitro model system based on a commercially available human neural progenitor cell line, ReNCell. We have engineered multiple distinct ReNCell lines to mimic specific genetic alterations associated with RTT, providing a robust platform for mechanistic studies and drug screening. This cell line carries a point mutation in the MECP2 gene (R133C), a common mutation in RTT patients, which alters the function of the MeCP2 protein. The model will allow us to study the impact of this mutation on neural development and function at a cellular level, providing insights into the disease's neuropathology.
2024-11-17 | E-MTAB-14046 | biostudies-arrayexpress
Project description:Rett Syndrome (RTT) is a severe neurological disorder predominantly affecting females, caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. Understanding the pathophysiology of RTT at a cellular and molecular level is crucial for the development of targeted therapies. Our project aims to dissect the molecular underpinnings of RTT using a novel in vitro model system based on a commercially available human neural progenitor cell line, ReNCell. We have engineered multiple distinct ReNCell lines to mimic specific genetic alterations associated with RTT, providing a robust platform for mechanistic studies and drug screening. This cell line is a complete knockout of MECP2, serving as a model to investigate the effects of total loss of MeCP2 function. This model helps in understanding the full spectrum of MeCP2's roles in neural development and maintenance, and in identifying compensatory mechanisms that could be targeted therapeutically. We capture the progenitor state (0 days), and differentiation states at 3, 7, 14, 21 and 30 days for both the MECP2 knockut and the corresponding wildtype.
2024-11-17 | E-MTAB-14047 | biostudies-arrayexpress
Project description:Single cell transcriptomic analysis of mutant MECP2 human neural cells over a time-course of in vitro differentiation
| PRJEB75396 | ENA
Project description:Rett Syndrome (RTT), a human neurodevelopmental disorder characterized by severe cognitive and motor impairments, is caused by dysfunction of the conserved transcriptional regulator Methyl-CpG-binding protein 2 (MECP2). Genetic analyses in mouse Mecp2 mutants, which exhibit key features of human RTT, have been essential for deciphering the mechanisms of MeCP2 function; nonetheless, our understanding of these complex mechanisms is incomplete. Zebrafish mecp2 mutants exhibit mild behavioral deficits but have not been analyzed in depth. Here we combine transcriptomic and behavioral assays to assess baseline and stimulus-evoked motor responses and sensory filtering in zebrafish mecp2 mutants from 5-7 days post-fertilization (dpf). We show that zebrafish mecp2 function is dispensable for gross movement, acoustic startle response, and sensory filtering (habituation and sensorimotor gating), and reveal a previously unknown role for mecp2 in behavioral responses to visual stimuli. RNA-seq analysis identified a large gene set that requires mecp2 function for correct transcription at 4 dpf, and pathway analysis revealed several pathways that require MeCP2 function in both zebrafish and mammals. These findings show that MeCP2’s function as a transcriptional regulator is conserved across vertebrates and supports using zebrafish to complement mouse modeling in elucidating these conserved mechanisms.
2024-01-01 | GSE252307 | GEO
Project description:Background MeCP2, methyl-CpG-binding protein 2, binds to methylated cytosines at CpG dinucleotides, as well as to unmethylated DNA, and affects chromatin condensation. MECP2 mutations in females lead to Rett syndrome, a neurological disorder characterized by developmental stagnation and regression, loss of purposeful hand use and speech, stereotypic hand movements, deceleration of brain growth, autonomic dysfunction and seizures. Most mutations occur de novo during spermatogenesis. Located at Xq28, MECP2 is subject to X inactivation, and affected females are mosaic. Rare hemizygous males suffer from a severe congenital encephalopathy. Methods To identify pathways mis-regulated by MeCP2 deficiency, microarray-based global gene expression studies were carried out on cerebellum of Mecp2 mutant mice. We compared transcript levels in mutant/wildtype male sibs of two different MeCP2-deficient mouse models at 2, 4 and 8 weeks of age. Increased transcript levels were evaluated by real-time quantitative RT-PCR. Chromatin immunoprecipitation assays were used to document in vivo MeCP2 binding to promoter regions of candidate target genes. Results In the mutants, several hundred genes showed altered expression levels. Twice as many were increased than decreased, and only 27 genes were differentially expressed at more than one time point. The number of misregulated genes was 30% lower in mice with an exon 3 deletion (Mecp2tm1.1Jae) than in mice with a larger deletion (Mecp2tm1.1Bird). Between the mutants, few misregulated genes overlapped at each time point. Real-time quantitative RT-PCR assays validated increased transcript levels for four genes: Irak1, interleukin-1 receptor-associated kinase 1; Fxyd1, phospholemman, associated with Na, K-ATPase; Reln, encoding reelin, an extracellular signaling molecule essential for neuronal lamination and synaptic plasticity; and Gtl2/Meg3, an imprinted maternally expressed non-translated RNA that serves as a host gene for C/D box snoRNAs and microRNAs. Chromatin immunoprecipitation assays documented in vivo MeCP2 binding to promoter regions of Fxyd1, Reln, and Gtl2. Conclusions Transcriptional profiling of cerebellum failed to detect significant global changes in Mecp2-mutant mice. Increased transcript levels of Irak1, Fxyd1, Reln, and Gtl2 may contribute to the neuronal dysfunction in MeCP2-deficient mice and individuals with Rett syndrome. Our data provide testable hypotheses for future studies of the regulatory or signaling pathways that these genes act on.
2007-07-17 | E-SMDB-4087 | biostudies-arrayexpress
Project description:NEAT1-Mediated Regulation in Rett Syndrome
| PRJEB81894 | ENA
Project description:The long noncoding RNA (lncRNA) Nuclear Enriched Abundant Transcript 1 (NEAT1) is a lncRNA involved in a variety of human cancers and diseases. The human NEAT1 gene produces two distinct isoforms, NEAT1 Long and NEAT1 Short, through alternative 3’ end formation. NEAT1 Long is an essential factor for nuclear paraspeckle formation, while the role of NEAT1 Short is poorly understood. Previous studies have often failed to distinctly detect the two NEAT1 isoforms and reported controversial NEAT1 dysregulation. Moreover, the molecular mechanisms which underlie the dysregulation of NEAT1 isoforms and their functional importance in tumorigenesis remain poorly characterized. We investigated whether usage of the proximal polyadenylation site (PAS) within the NEAT1 transcript is regulated to govern the biogenesis of NEAT1 isoforms in human glioma cells. We found differential dysregulation of NEAT1 isoforms in patient-derived human glioblastoma multiforme (GBM) stem cells. We further show deletion of the NEAT1 PAS reduced NEAT1 Short and increased NEAT1 Long. We identified the RNA binding protein QKI, a risk factor for glioma, facilitates the utilization of the NEAT1 PAS. We present evidence indicating the imbalance of NEAT1 isoforms correlates with transcriptomic pathway changes. We propose QKI-5 regulates NEAT1 isoform biogenesis through modulating the NEAT1 PAS in human glioma cells.
2024-10-01 | GSE262598 | GEO
Project description:This dataset comprises 15 raw AP-MS files, each with an associated peak list, captured using a Vanquish Neo nanoLC system in tandem with an Orbitrap Eclipse mass spectrometer. To generate MECP2 hESC-reporter lines for wild type (WT) and various mutations of MECP2, including R133C, R168X, and R270X, we first used CRISPR/Cas9 to create MECP2 alleles carrying the green fluorescent protein (GFP) sequences in the endogenous gene. The R133C mutation was then introduced into the WT MECP2-GFP reporter line. Mutations R133C, R168X, and R270X are recognized as loss-of-function variants in MECP2 and are also identified as primary Rett syndrome-causing mutations. For efficient neuronal differentiation, a doxycycline (DOX)-responsive NGN2 construct was incorporated at their AAVS1 safe harbor locus. Upon the addition of DOX, homogenous populations of neurons were generated within three weeks from those four MECP2 hESC-reporter lines. Subsequently, GFP-pull down assay and AP-MS were performed using these WT MECP2-GFP neurons along with R133C-, R168X-, and R270X-mutant MECP2-GFP reporter neurons. Neurons expressing only the GFP tag served as a negative control. AP-MS analysis identified proteins interacting differently between WT and mutant MECP2 within human neurons.
2023-10-27 | MSV000093210 | MassIVE