Enhancer distance matters: forced linear recruitment of a super-enhancer strongly reactivates the developmentally silenced fetal HBG globin genes [CutnRun]
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ABSTRACT:
PROVIDER: PRJNA1144489 | ENA |
REPOSITORIES: ENA
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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
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:Enhancer distance matters: forced linear recruitment of a super-enhancer strongly reactivates the developmentally silenced fetal HBG globin genes [ATAC]
| PRJNA1144488 | ENA
Project description:The most common genetic disorders worldwide are the β-hemoglobinopathies, including sickle cell disease and β-thalassemia caused by HBB gene mutations. Five functional β-like globin genes on chromosome 11 are expressed during development in a tissue and stage-specific manner. The HBG genes encoding g-globin polypeptide chains to form fetal hemoglobin (HbF), undergo gene silencing during the first year of life. Our study aimed to characterize an upstream DNA regulatory region that can be targeted for HBG reactivation during adult development. We investigated the intergenic region between HBE1 and HBG2 which we termed the fetal chromatin domain (FCD) to define its role in globin gene regulation. Using normal human CD34+ stem cells to establish erythroid progenitors, we characterized the binding of different transcription factors to the FCD, which facilitated long-range chromatin looping between the FCD and locus control region and HBG. Using CRISPR/Cas9 genome editing technology, the FCD was knocked out in KU812 and CD34+ stem cells. We observed enhanced HBG transcription and HbF expression in both lines. For FCD knockout CD34+ stem cells, flow cytometry showed increased HbF positive cells and HbF expression and enhanced long-range interactions between the locus control and HBG region. Single cell RNA-sequencing revealed normal erythropoiesis in the FCD-KO line and partial reversal of HBG silencing. These results support a putative silencer role of the FCD in regulating HBG transcription and HbF expression.
2025-11-26 | GSE311528 | 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 | 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:We performed epigenomic profiling to evaluate the effects of arachidonic acid (AA) on intestinal stem cell regeneration via CutnRun and found that AA treatment led to global changes in methylation status of genes that are associated with stem cell reprogramming.
2025-11-01 | GSE246876 | GEO