A modular CRISPRa system for molecular therapy of FOXG1 syndrome
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1151651 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR30352804_1.fastq.gz | Fastqsanger.gz | |||
| SRR30352804_2.fastq.gz | Fastqsanger.gz | |||
| SRR30352805_1.fastq.gz | Fastqsanger.gz | |||
| SRR30352805_2.fastq.gz | Fastqsanger.gz | |||
| SRR30352806_1.fastq.gz | Fastqsanger.gz |
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Project description:To date, there is an unmet need for novel approaches that address haploinsufficiency disorders in a targeted and robust manner. Here, we developed a novel, modular tool for transcriptional regulation of genes involved in neurodevelopmental disease in the absence of creating double-stranded breaks. We utilized this novel platform technology to identify sgRNAs that efficiently regulate expression of the intellectual disability gene FOXG1 that utilizes an optimized protein intein system. Using a modular assembly of CRISPR trans-activators, including single and dual VP64 dCas9 fusion proteins, sgRNA-mediated MS2 p65 HSF (MPH) recruitment or the addition of the VPR tripartite effector (VP64, p65, Rta) can induce dynamic gene regulation. We further increased mRNA levels of FOXG1 using duplexed sgRNAs paired with different combinations of transcriptional activators. We demonstrate that FOXG1 gene upregulation results in a significant increase in FoxG1 protein translation. Following treatment of mutant FOXG1 neural stem cells with the CRISPR activators, we observed a significant increase in FOXG1 expression with no-off targets as determined by in silico sgRNA binding prediction paired with RNA-sequencing. This approach holds great promise as a modular platform technology that is readily adapted for gene therapy delivery for those affected by rare genetic conditions.
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Project description:Rett syndrome is a complex neurodevelopmental disorder that is mainly caused by mutations in MECP2. However, mutations in FOXG1 cause a less frequent non-congenital form called atypical Rett syndrome. FOXG1 is a key transcription factor implicated in forebrain development, where it maintains the balance between progenitor proliferation and neuronal differentiation. Using SILAC based quantitative proteomics and genome-wide small RNA sequencing, we identified that FOXG1 interacts with the ATP-dependent RNA helicase, DDX5/p68 and controls the biogenesis of miRNAs. Both, FOXG1 and DDX5 bind to the miR200b/a/429 primary transcript and associate with the microprocessor complex, whereby DDX5 recruits FOXG1 to DROSHA. In vivo and in vitro experiments show that both FOXG1 and DDX5 are necessary for effective maturation of miR200b/a/429. RNAseq analyses of Foxg1-heterozygote hippocampi and miR200b/a/429 overexpressing Neuro-2a cells revealed that the cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) is a target of miR200 in neural cells. Since it is known that PRKAR2B inhibits postsynaptic functions by attenuating protein kinase A (PKA) activity, increased PRKAR2B levels may contribute to neuronal dysfunctions in FOXG1 Rett syndrome.
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Project description:Next-generation sequencing facilitates quantitative analysis of the transcriptomes of FOXG1 100% dosage GABA interneurons, FOXG1 60% dosage GABA interneurons, FOXG1 30% dosage GABA interneurons, and FOXG1 0% dosage GABA interneurons derived from human embryonic stem cells. We report a genetic manipulation system that enable precise dosage control of FOXG1 protein in human pluripotent stem cells (hPSCs). Using this system, we explored how the various reduced dosage affect human ventrol GABA interneuron development. We employed RNA seq on hPSC-derived GABA interneurons (day 60) to invest the expression pattern under different FOXG1 dosage conditions. RNA-Seq on GABA interneurons (Day 60) indicates that compared to the FOXG1 100% group, variable insufficiency of FOXG1 produces more than 1000 differently expressed genes (DEGs), and more DEGs in the group with less FOXG1 dosage. Heat map on Pearson Correlation indicates that groups with more discriminated FOXG1 exhibit much weaker correlation. Venn diagram reveals that each group has a set of distinct DEGs, suggesting that each FOXG1 protein dosage could results in different expression pattern during differentiation. The DEGs can be divided into two clusters, with one showing dosage-dependent regulation by FOXG1 and the other one typical binary. Key regulatory genes for GABA interneuron induction (NKX2-1, NKX6-2, GAD1, etc.) and for functional GABAergic-specific synapse formation (GABBR1, GABRA1, GABRB1, GABRG1, GABRQ, SHANK1, etc.) are down regulated along with reduction of FOXG1 protein.
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Project description:The integrative and multi-omics view of changes upon FOXG1 reduction reveals an unprecedented multimodality of FOXG1 functions converging on neuronal maturation, fueling novel therapeutic options based on epigenetic drugs to alleviate, at least in part, neuronal dysfunctions. This RNA-seq dataset is part of tis multimodal invetigation of FOXG1 functions.
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2019-09-22 | GSE104169 | GEO
Project description:As part of our multimodal investigation fo FOXG1 functions at the chromatin, we report that there are both increase and decrease in chromatin accessibility upon reduced levels of FOXG1 in pirmary hippocampal neurons. The data presented in this study enlighten for the first time in a comprehensive manner diverse and multi-modal functions of FOXG1 at the chromatin level in mature neurons. Despite FOXG1 being recognized as a key transcription factor for telencephalic development and neuronal function, insights into the mechanism underlying transcriptional regulation are sparse. We here show that (i) FOXG1 acts both as repressor and activator, (ii) localizes predominantly to enhancer regions, (iii) alters the epigenetic landscape, (iv) affects directly HDAC functions, and (v) acts in concert with NEUROD1 to instruct transcriptional programs necessary for axono- and synaptogenesis.
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Project description:Disruptions in gene expression programs during nervous system development?affecting both transcriptional and post-transcriptional regulation?are implicated in a range of neurodevelopmental disorders. Among these, FOXG1 syndrome remains poorly understood, particularly with respect to the mechanistic role of its namesake gene, FOXG1. In this study, we performed single-cell RNA sequencing (scRNA-seq) on day 105 (d105) cerebral organoids derived from healthy controls as well as from iPSCs carrying FOXG1^del and FOXG1^c.460dupG mutations, to investigate cell type?specific gene expression changes.
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Project description:We report that FOXG1 localises at promoter, distal intergenic and intronic regions in vitro.The data presented in this study enlighten for the first time in a comprehensive manner diverse and multi-modal functions of FOXG1 at the chromatin level in mature neurons. Despite FOXG1 being recognized as a key transcription factor for telencephalic development and neuronal function, insights into the mechanism underlying transcriptional regulation are sparse. We here show that (i) FOXG1 acts both as repressor and activator, (ii) localizes predominantly to enhancer regions, (iii) alters the epigenetic landscape, (iv) affects directly HDAC functions, and (v) acts in concert with NEUROD1 to instruct transcriptional programs necessary for axono- and synaptogenesis.
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Project description:FOXG1 syndrome is a developmental encephalopathy with a high phenotypic variability, which results from FOXG1 mutations. However, the upstream transcriptional regulation of Foxg1 expression remains unclear. Here we report that both deficiency and overexpression of Men1 (protein: menin, a pathogenic gene of MEN1 syndrome) result in autism-like behaviors, including social defects, increased repetitive behaviors and cognition impairments. We employed multifaceted transcriptome analyses and found that Foxg1 signaling is mostly altered in Men1 deficiency mice, through its regulation over Alpha Thalassemia/Mental Retardation Syndrome X-Linked (Atrx) factor. Atrx recruits menin to bind to the transcriptional start region of Foxg1 and mediates the regulation of Foxg1 expression by H3K4me3 modification. Notably, the described changes in menin deficient mice were rescued by over-expression of Foxg1, leading to normalized spine growth and hippocampal synaptic plasticity. Collectively, these results indicate a putative role of menin in maintaining Foxg1 expression, and menin signaling may serve as Foxg1-related encephalopathy therapeutic targets.
2024-07-03 | GSE261274 | GEO