Removal of promoter DNA methylation by epigenome editing reverses HBG silencing [CUT&RUN]
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1198350 | ENA |
REPOSITORIES: ENA
Similar Datasets
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 | 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 | GSE284289 | GEO
Project description:Removal of promoter DNA methylation by epigenome editing reverses HBG silencing [RNA-seq]
| PRJNA1198349 | ENA
Project description:Removal of promoter DNA methylation by epigenome editing reverses HBG silencing [ATAC-seq]
| PRJNA1198351 | ENA
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
| PRJNA656903 | ENA
Project description:Expanded CAG/CTG repeat disorders affect over 1 in 2500 individuals worldwide. Potential therapeutic avenues include gene silencing and modulation of repeat instability. However, there are major mechanistic gaps in our understanding of these processes, which prevent the rational design of an efficient treatment. To address this, we developed a novel system, ParB/ANCHOR-mediated Inducible Targeting (PInT), in which any protein can be recruited at will to a GFP reporter containing an expanded CAG/CTG repeat. Previous studies have implicated the histone deacetylase HDAC5 and the DNA methyltransferase DNMT1 as modulators of repeat instability via mechanisms that are not fully understood. Using PInT, we found no evidence that HDAC5 or DNMT1 modulate repeat instability upon targeting to the expanded repeat, suggesting that their effect is independent of local chromatin structure. Unexpectedly, we found that expanded CAG/CTG repeats reduce the effectiveness of gene silencing mediated by targeting HDAC5 and DNMT1. The repeat-length effect in gene silencing by HDAC5 was abolished by a small molecule inhibitor of HDAC3. Our results have important implications on the design of epigenome editing approaches for expanded CAG/CTG repeat disorders. PInT is a versatile synthetic system to study the effect of any sequence of interest on epigenome editing.
| S-EPMC8825355 | biostudies-literature
Project description:Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.
| S-EPMC4666778 | biostudies-literature
Project description:Mounting evidence has called into question our understanding of the role that the central dogma of molecular biology plays in human pathology. The conventional view that elucidating the mechanisms for translating genes into proteins can account for a panoply of diseases has proven incomplete. Landmark studies point to epigenetics as a missing piece of the puzzle. However, technological limitations have hindered the study of specific roles for histone post-translational modifications, DNA modifications, and non-coding RNAs in regulation of the epigenome and chromatin structure. This feature highlights CRISPR systems, including CRISPR-Cas9, as novel tools for targeted epigenome editing. It summarizes recent developments in the field, including integration of optogenetic and functional genomic approaches to explore new therapeutic opportunities, and underscores the importance of mitigating current limitations in the field. This comprehensive, analytical assessment identifies current research gaps, forecasts future research opportunities, and argues that as epigenome editing technologies mature, overcoming critical challenges in delivery, specificity, and fidelity should clear the path to bring these technologies into the clinic.
| S-EPMC5168826 | biostudies-literature