Removal of promoter DNA methylation by epigenome editing reverses HBG silencing [RNA-seq]
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ABSTRACT:
PROVIDER: PRJNA1198349 | ENA |
REPOSITORIES: ENA
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Project description:Removal of promoter DNA methylation by epigenome editing reverses HBG silencing [CUT&RUN]
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Project description:Removal of promoter DNA methylation by epigenome editing reverses HBG silencing [ATAC-seq]
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Project description:Beta-hemoglobinopathies caused by mutations in adult-expressed HBB can be treated by re-activating the adjacent paralogous genes HBG1 and HBG2 (HBG), which are normally silenced perinatally. Although HBG expression is induced by global demethylating drugs, their mechanism is poorly understood and toxicity limits their use. We identified the DNMT1-associated maintenance methylation protein UHRF1 as a mediator of HBG repression in a genome-wide screen. Loss of UHRF1 in the adult-type erythroid cell line HUDEP2 caused global demethylation and HBG activation that was reversed upon localized promoter re-methylation. Conversely, targeted demethylation of the HBG promoters activated their genes in HUDEP2 or primary CD34+ cell-derived erythroblasts. Mutation of MBD2, a CpG-methylation reading component of the NuRD complex, to impair methylation sensitivity recapitulated the effects of promoter demethylation. Our studies demonstrate that localized CpG-methylation at the HBG promoters facilitates developmental silencing and identify a targeted therapeutic approach for b-hemoglobinopathies via epigenomic editing.
2025-06-24 | GSE284290 | GEO
Project description:Beta-hemoglobinopathies caused by mutations in adult-expressed HBB can be treated by re-activating the adjacent paralogous genes HBG1 and HBG2 (HBG), which are normally silenced perinatally. Although HBG expression is induced by global demethylating drugs, their mechanism is poorly understood and toxicity limits their use. We identified the DNMT1-associated maintenance methylation protein UHRF1 as a mediator of HBG repression in a genome-wide screen. Loss of UHRF1 in the adult-type erythroid cell line HUDEP2 caused global demethylation and HBG activation that was reversed upon localized promoter re-methylation. Conversely, targeted demethylation of the HBG promoters activated their genes in HUDEP2 or primary CD34+ cell-derived erythroblasts. Mutation of MBD2, a CpG-methylation reading component of the NuRD complex, to impair methylation sensitivity recapitulated the effects of promoter demethylation. Our studies demonstrate that localized CpG-methylation at the HBG promoters facilitates developmental silencing and identify a targeted therapeutic approach for b-hemoglobinopathies via epigenomic editing.
2025-06-24 | GSE284291 | GEO
Project description:Beta-hemoglobinopathies caused by mutations in adult-expressed HBB can be treated by re-activating the adjacent paralogous genes HBG1 and HBG2 (HBG), which are normally silenced perinatally. Although HBG expression is induced by global demethylating drugs, their mechanism is poorly understood and toxicity limits their use. We identified the DNMT1-associated maintenance methylation protein UHRF1 as a mediator of HBG repression in a genome-wide screen. Loss of UHRF1 in the adult-type erythroid cell line HUDEP2 caused global demethylation and HBG activation that was reversed upon localized promoter re-methylation. Conversely, targeted demethylation of the HBG promoters activated their genes in HUDEP2 or primary CD34+ cell-derived erythroblasts. Mutation of MBD2, a CpG-methylation reading component of the NuRD complex, to impair methylation sensitivity recapitulated the effects of promoter demethylation. Our studies demonstrate that localized CpG-methylation at the HBG promoters facilitates developmental silencing and identify a targeted therapeutic approach for b-hemoglobinopathies via epigenomic editing.
2025-06-24 | GSE284289 | GEO
Project description:Efficiency of HBG gene activation by the Cas12f/cgRNA system. In a doxycycline-inducible dCas12f-VPR knock-in (KI) HEK293T cell line, transiently transfected plasmids encoding U6, linear, and circular gRNAs targeting HBG1, and cgRNAs targeting mNeonGreen (negative control, NC) to activate HBG gene expression. RNA-seq analysis showed that cgRNAs exhibited increased efficiency in the target gene HBG1 and slight lower specificity when compared with U6 and linear gRNAs.
2024-03-12 | GSE261105 | GEO
Project description:Heterorhabditis indica isolate:NBAII HBG Genome sequencing
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Project description:Epigenetic aberration is one of the major driving factors in human cancer, often leading to acquired resistance to chemotherapies. Various chemical epigenetic modulators have been reported. Nonetheless, outcomes from animal models and clinical trials have underscored the substantial setbacks attributed to pronounced on- and off-target toxicities. To address these challenges, CRISPR/dCas9 technology emerges as a potent tool for precise epigenetic regulation. This relies on the co-expression of exogenous epigenetic modulator proteins, presenting technical challenges during preparation and delivery, as well as potential undesirable side effects. Recently, our research demonstrated that Cas9 tagged with the FCPF-peptide motif could be recognized by perfluoro biphenyl (PFB) derivatives. Here, we integrated the FCPF-tag into dCas9 and established a chemically inducible platform for epigenome editing, called Chem-CRISPR/dCas9FCPF. We designed a series of chemical inhibitor-PFB conjugates targeting various epigenetic modulator proteins. Focusing on JQ1, a BRD4 inhibitor, we demonstrate that c-MYC-sgRNA-guided JQ1-PFB specifically inhibits BRD4 in close proximity to the c-MYC promoter/enhancer, thereby effectively repressing the intricate transcription networks orchestrated by c-MYC. In conclusion, our Chem-CRISPR/dCas9FCPF platform presents a promising alternative to conventional fusion protein systems. It replaces exogenous epigenetic modulator proteins with chemical inhibitors, offering a highly efficient and precise approach to epigenome editing.
2025-05-06 | PXD048673 | Pride
Project description:The human genome contains regulatory DNA elements (enhancers, silencers, insulators) that can recruit proteins to activate or repress transcription of distal genes. Interspersed are DNA intervals without any known coding or regulatory capacity. Here, we show that these intrinsically non-regulatory DNA stretches can allow gene silencing by keeping enhancers at distance. During development, the fetal globin HBG genes recruit activated repressor proteins to their promoters. This enables the HBG genes to silence and ignore the distal super-enhancer, which subsequently acts on and activates the downstream adult HBD and HBB genes. We found that forced linear recruitment of the super-enhancer, bringing it immediately upstream of the otherwise intact HBG gene promoters, results in significant HBG reactivation and partial HBB inactivation in adult erythroid cells and ex vivo differentiated human hematopoietic stem and progenitor cells. Forced HBG reactivation is accomplished by CRISPR-Cas9 deletions of the intervening 25 kilobases sequence interval. Additionally, it can be achieved by inversions that leave this interval intact and proximal, but no longer situated between the enhancer and HBG genes. We conclude that the HBG genes must be at substantial linear distance to bypass activation by the super-enhancer and activate their promoter-encoded silencing program in adulthood. Overall, this assigns a functional role to seemingly non-regulatory segments in our genome: by providing linear separation they may support genes to autonomously control their transcriptional response to distal enhancers.
2025-08-05 | GSE274029 | GEO
Project description:The human genome contains regulatory DNA elements (enhancers, silencers, insulators) that can recruit proteins to activate or repress transcription of distal genes. Interspersed are DNA intervals without any known coding or regulatory capacity. Here, we show that these intrinsically non-regulatory DNA stretches can allow gene silencing by keeping enhancers at distance. During development, the fetal globin HBG genes recruit activated repressor proteins to their promoters. This enables the HBG genes to silence and ignore the distal super-enhancer, which subsequently acts on and activates the downstream adult HBD and HBB genes. We found that forced linear recruitment of the super-enhancer, bringing it immediately upstream of the otherwise intact HBG gene promoters, results in significant HBG reactivation and partial HBB inactivation in adult erythroid cells and ex vivo differentiated human hematopoietic stem and progenitor cells. Forced HBG reactivation is accomplished by CRISPR-Cas9 deletions of the intervening 25 kilobases sequence interval. Additionally, it can be achieved by inversions that leave this interval intact and proximal, but no longer situated between the enhancer and HBG genes. We conclude that the HBG genes must be at substantial linear distance to bypass activation by the super-enhancer and activate their promoter-encoded silencing program in adulthood. Overall, this assigns a functional role to seemingly non-regulatory segments in our genome: by providing linear separation they may support genes to autonomously control their transcriptional response to distal enhancers.
2025-08-05 | GSE274030 | GEO