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DNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library in haploid human embryonic stem cells transfected with a methylated construct containing the FXS-related mutation upstream to an EGFP reporter gene

ABSTRACT: The primary mechanism causing Fragile X syndrome (FXS) is the expansion and silencing of a repetitive CGG sequence in the 5’-UTR of the FMR1 gene. To identify novel epigenetic pathways involved in FXS pathogenesis, we have established a model system for FMR1 silencing using a construct containing the FXS-related FMR1 CGG expansion upstream to an EGFP reporter gene. This construct was methylated in vitro and introduced into a genome-wide loss-of-function (LOF) library established in haploid human pluripotent stem cells. Library cells were then sorted to GFP-positive and GFP-negative populations. The analysis of the changes in abundance of mutants between the GFP-positive and GFP-negative populations has yielded a list of candidate genes whose functional loss reversed the methylation-induced silencing of the expanded FMR1 5'-UTR sequence.

ORGANISM(S): Homo sapiens

PROVIDER: GSE182551 | GEO | 2022/06/01

REPOSITORIES: GEO

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RNAseq analysis of CRISPR/Cas9-based perturbation of DNMT1 in FXS induced pluripotent stem cells

Project description:The primary mechanism causing Fragile X syndrome (FXS) is the expansion and silencing of a repetitive CGG sequence in the 5’-UTR of the FMR1 gene. Previous work has demonstrated that chemical disruption of DNA methylation induces FMR1 expression in FXS-iPSCs. To further explore the maintenance of DNA methylation in the FMR1 locus, and to test the possibility of FMR1 reactivation using gene targeting of a single epigenetic factor, we performed a global transcriptional analysis of FXS-iPSCs infected with a lentiviral construct containing Cas9 and sgRNA targeting DNMT1.

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Rescue of Fragile X syndrome neurons by DNA methylation editing of the FMR1 gene [RNA-seq]

Project description:Fragile X syndrome (FXS), the most common genetic form of intellectual disability in male, is caused by silencing of the FMR1 gene by hypermethylation of the CGG expansion mutation in the 5’UTR region of FMR1 in FXS patients. Here, we applied recently developed DNA methylation editing tools to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/sgRNA switched the heterochromatin status of the upstream FMR1 promoter to an active chromatin state restoring a persistent expression of FMR1 in FXS iPSCs. Neurons derived from methylation edited FXS iPSCs rescued the electrophysiological abnormalities and restored a wild-type phenotype upon the mutant neurons. FMR1 expression in edited neurons was maintained in vivo after engrafting into the mouse brain. Finally, demethylation of the CGG repeats in post-mitotic FXS neurons also reactivated FMR1. Our data establish demethylation of the CGG expansion is sufficient for FMR1 reactivation, suggesting potential therapeutic strategies for FXS.

2018-02-16 | GSE108498 | GEO

Rescue of Fragile X syndrome neurons by DNA methylation editing of the FMR1 gene [methylation]

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Rescue of Fragile X syndrome by DNA methylation editing of the FMR1 [ChIP-seq II]

Project description:Fragile X syndrome (FXS), the most common genetic form of intellectual disability in male, is caused by the silence of FMR1. Hypermethylation of the CGG expansion mutation in the 5’UTR region of FMR1 in FXS patients was thought to epigenetically silence FMR1. Here, we applied our previously developed DNA methylation editing tool to reverse this hypermethylation event. Targeted demethylation of the CGG expansion by dCas9-Tet1/gRNA switched the heterochromatin status of the FMR1 promoter to an active chromatin status and subsequently restored FMR1 expression in FXS iPSCs. Neurons derived from methylation edited FXS iPSCs showed a similar electrophysiological property as wild-type neurons, and maintained FMR1 expression for months after engrafting into the mouse brain. Reactivation of FMR1 can be achieved in FXS neurons with demethylation of the CGG expansion. Lastly, we showed that targeted demethylation of the FMR1 promoter can reactivate FMR1 as well suggesting potential therapeutic approaches for FXS.

2018-02-16 | GSE102684 | GEO

Rescue of Fragile X syndrome by DNA methylation editing of the FMR1 [ChIP-seq]

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Genome-wide analysis identifies aberrant methylation in Fragile X syndrome is specific to the FMR1 locus

Project description:Fragile X syndrome (FXS) is a common form of inherited intellectual disability and is caused by an expansion of CGG repeats located in the 5Õ untranslated region (UTR) of the FMR1 gene, leading to hypermethylation and silencing of this locus. While the dramatic increase in DNA methylation (DNAm) of the FMR1 full mutation allele is well documented, the extent that these changes affect DNAm throughout the entire gene and the rest of the genome remains unexplored. Here, we examine the genome-wide methylation in peripheral blood (N = 9) as well as induced pluripotent stem cells (iPSCs; N = 10) from FXS individuals and controls (N = 53 and 9, respectively) and find the expected significant DNAm differences in the FMR1 promoter and 5Õ UTR, but also that these changes inversely persist throughout the FMR1 gene body. Importantly, we find there are no additional differential methylated loci (DML) throughout the remainder of the genome, indicating that the aberrant methylation of the FMR1 in FXS is locus-specific and does not change DNAm genome-wide. This study provides a comprehensive methylation profile of FXS and refines mechanistic considerations of FMR1 silencing. A total of 62 blood (53 controls + 9 Fragile X) samples analyzed using a linear regression model.

2013-04-15 | E-GEOD-41273 | biostudies-arrayexpress

Site-specific R-loop formation induces CGG repeat contraction and Fragile X gene reactivation

Project description:Fragile X syndrome (FXS) is a neurodevelopmental disorder and a leading cause of intellectual disability. In FXS, the neuronal regulator, FMR1, is epigenetically silenced by a CGG repeat expansion. Here, we investigate conditions under which repeat expansion and gene silencing could be reversed. Surprisingly, inducing formation of R-loops (3-stranded RNA-DNA structures) within FMR1 is sufficient to initiate CGG contraction, promoter demethylation, and FMR1 reactivation. Recruiting RNaseH degrades the R-loop and abolishes the response. Targeting the nascent mRNA for degradation also eliminates the response. Thus, we have identified an exogenous nuclease-free method of contracting CGG repeats and reversing FMR1 silencing. We propose that DNA demethylation, new transcription, and R-loop formation engage in a feed-forward cycle to contract CGG repeats and reactivate FMR1.

2023-05-19 | GSE184580 | GEO

Reversion of FMR1 methylation and silencing by editing the triplet repeats in Fragile-X iPSC-derived neurons

Project description:Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from a CGG repeat expansion in the fragile X mental retardation 1 (FMR1) gene. Here we report a strategy for CGG repeat correction using CRISPR/Cas9 for targeted deletion in both embryonic stem cells and induced pluripotent stem cells derived from FXS patients. Following gene correction in FXS induced pluripotent stem cells, FMR1 expression was restored and sustained in neural precursor cells and mature neurons. Strikingly, after removal of the CGG repeats, the upstream CpG island of the FMR1 promoter showed extensive demethylation, an open chromatin state, and transcription initiation. These results suggest a silencing maintenance mechanism for the FMR1 promoter that is dependent on the existence of the CGG repeat expansion. Our strategy for deletion of trinucleotide repeats provides further insights into the molecular mechanisms of FXS and future therapies of trinucleotide repeat disorders.

2015-08-18 | GSE72114 | GEO

Genome-wide analysis identifies aberrant methylation in Fragile X syndrome is specific to the FMR1 locus

2013-04-15 | GSE41273 | GEO

Exome-sequencing of two UFM individuals and their Fragile X family members.

Project description:Fragile X syndrome (FXS) is a monogenic neurodevelopmental disease often caused by a CGG triplet expansion in the 5’UTR of the FMR1 gene, which results in DNA methylation of the FMR1 promoter and its transcriptional silencing. Exceptional healthy individuals named "unmethylated full mutation (UFM)" have been identified in the families of FX patients. They carry an expanded CGG repeat, but show normal FMR1 epigenetic configuration and expression. In this study we performed exome-sequencing of two UFM individuals and their family members to identify possible genetic mutations responsible for the escape of silencing in the unmethylated full mutation (UFM) phenotype.

| EGAS00001001737 | EGA

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