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Artificial escape from XCI by DNA methylation editing of the CDKL5 gene [RNA-seq]

ABSTRACT: A significant number of X-linked genes escape from X-chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we sought to create an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels above 60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X-chromosome inactivation. Understanding the epigenetics associated with escape from X-chromosome inactivation has potential for those suffering from X-linked disorders.

ORGANISM(S): Homo sapiens

PROVIDER: GSE137663 | GEO | 2020/01/01

REPOSITORIES: GEO

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Artificial escape from XCI by DNA methylation editing of the CDKL5 gene [methylation array]

Project description:A significant number of X-linked genes escape from X-chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we sought to create an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels above 60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X-chromosome inactivation. Understanding the epigenetics associated with escape from X-chromosome inactivation has potential for those suffering from X-linked disorders.

2020-01-01 | GSE137541 | GEO

Multiplex Epigenome Editing of MECP2 to Rescue Rett Syndrome Neurons (WGBS)

Project description:Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by loss-of-function heterozygous mutations of MECP2. Reactivation of the silent wild-type MECP2 allele on the inactive X chromosome (Xi) represents a promising therapeutic opportunity for female RTT patients. Here, we applied a multiplex epigenome editing approach to reactivate MECP2 on Xi. Demethylation of the MECP2 promoter by dCas9-Tet1 with target sgRNA reactivated MECP2 on Xi in RTT hESCs without detectable off-target effects at the transcriptome level. Neurons derived from methylation edited RTT hESCs reversed the smaller soma size and electrophysiological abnormalities. Insulation of the methylation edited MECP2 locus in RTT neurons by dCpf1-CTCF with target crRNA stabilized MECP2 reactivation and rescued the RTT-related neuronal defects, providing a proof-of-concept study for multiplex epigenome editing to treat RTT.

2023-09-01 | GSE214549 | GEO

Multiplex Epigenome Editing of MECP2 to Rescue Rett Syndrome Neurons (ChIP-Seq)

2023-09-01 | GSE214547 | GEO

Multiplex Epigenome Editing of MECP2 to Rescue Rett Syndrome Neurons (RNA-Seq)

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Escape from X inactivation in mouse tissues (RNA-Seq)

Project description:X chromosome inactivation (XCI) silences most genes on one X chromosome in female mammals, but some genes escape XCI. To identify escape gene in vivo and to explore molecular mechanisms that regulate this process we analyzed the allele-specific expression and chromatin structure of X-linked genes in mouse tissues and cells with skewed XCI and distinguishable alleles based on single nucleotide polymorphisms. Using a new method to estimate allelic expression, we demonstrate a continuum between complete silencing and significant expression from the inactive X (Xi). Few genes (2-3%) escape XCI to a significant level and only a minority differs between mouse tissues, suggesting stringent silencing and escape controls. Allelic profiles of DNase I hypersensitivity and RNA polymerase II occupancy of genes on the Xi correlate with escape from XCI. Allelic binding profiles of the DNA binding protein CCCTC-binding factor (CTCF) in different cell types indicate that CTCF binding at the promoter correlates with escape. Importantly, CTCF binding at the boundary between escape and silenced domains may prevent the spreading of active escape chromatin into silenced domains. Examination of allelic expression in mouse hybrid tissues.

2015-02-20 | E-GEOD-59777 | biostudies-arrayexpress

Escape from X inactivation in mouse tissues (ChIP-Seq)

2015-02-20 | E-GEOD-59778 | biostudies-arrayexpress

Dynamic Erasure of Random X-Chromosome Inactivation during iPSC Reprogramming

Project description:Induction and reversal of chromatin silencing is critical for successful development, tissue homeostasis and the derivation of induced pluripotent stem cells (iPSCs). X-chromosome inactivation (XCI) and reactivation (XCR) in female cells represent chromosome-wide transitions between active and inactive chromatin states. While XCI has long been studied and provided important insights into gene regulation, the dynamics and mechanisms underlying the reversal of stable chromatin silencing of X-linked genes are much less understood. In this work, we use allele-resolution transcriptomic approaches to study XCR during mouse iPSC reprogramming in order to elucidate the timing and mechanisms of chromosome-wide reversal of gene silencing. Our findings reveal a sequential hierarchy of gene reactivation from the inactive X-chromosome (Xi). We show that gene reactivation initiates before the activation of late pluripotency genes and complete silencing of the long non-coding RNA (lncRNA) Xist, and is completed late in reprogramming. We reveal that the gene-specific timing of reactivation correlates with the genomic distance to genes that escape inactivation and with differential targeting by pluripotency transcription factors (TFs). We also show that histone deacetylases restrict XCR in reprogramming intermediates and that the severe hypoacetylation state of the Xi persists until late reprogramming stages. Therefore, randomly inactivated X-linked genes possess different degrees of epigenetic memory, the reversal of which may involve the combined action of chromatin regulators, pluripotency transcription factors and chromatin topology. Taken together, our study provides the chromosome-wide dynamics of gene reactivation from the randomly inactivated X-chromosome and a framework for understanding how transcription factors induce dynamic reversal of stable epigenetic memory by overcoming repressive chromatin barriers.

2019-02-08 | GSE126229 | GEO

Regulation of mouse X inactivation (ChIP-Seq)

2015-02-20 | GSE59778 | GEO

Escape from X inactivation in mouse tissues (RNA-Seq)

2015-02-20 | GSE59777 | GEO

Editing DNA methylation in the mammalian genome

Project description:Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice demonstrating their wide utility for functional studies of epigenetic regulation.

2016-09-23 | GSE83890 | GEO

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