Ontology highlight
ABSTRACT:
PROVIDER: PRJNA985399 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24965199.fastq.gz | Fastqsanger.gz |
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Project description:The mechanisms by which the fetal type b-globin-like genes HBG1 and HBG2 are silenced in adult erythroid precursor cells is a basic biology question in human development. Reversal of such mechanisms is beneficial for b hemoglobinopathies, such as sickle cell disease (SCD). A CRISPR-Cas9 genetic screen uncovered two members of the NFI transcription factor family – NFIA and NFIX – as novel HBG1/2 repressors. Both factors are expressed at elevated levels in adult erythroid cells, and their single or combined depletion revealed cooperativity in HBG1/2 regulation in cultured cells and human-to-mouse xenotransplant experiments, as well as in preventing sickling of SCD-derived erythroblasts. Genomic profiling, gene editing and in vitro binding assays demonstrated that the potent concerted activity of NFIA and NFIX factors is explained in part by their ability to activate expression of BCL11A, a known silencer of the HBG1/2 genes, and in part by repressing the HBG1/2 genes via two direct binding sites. NFI factors emerge as versatile regulators of the fetal-to-adult switch in b-globin production.
2022-03-23 | GSE180860 | GEO
Project description:The mechanisms by which the fetal type b-globin-like genes HBG1 and HBG2 are silenced in adult erythroid precursor cells is a basic biology question in human development. Reversal of such mechanisms is beneficial for b hemoglobinopathies, such as sickle cell disease (SCD). A CRISPR-Cas9 genetic screen uncovered two members of the NFI transcription factor family – NFIA and NFIX – as novel HBG1/2 repressors. Both factors are expressed at elevated levels in adult erythroid cells, and their single or combined depletion revealed cooperativity in HBG1/2 regulation in cultured cells and human-to-mouse xenotransplant experiments, as well as in preventing sickling of SCD-derived erythroblasts. Genomic profiling, gene editing and in vitro binding assays demonstrated that the potent concerted activity of NFIA and NFIX factors is explained in part by their ability to activate expression of BCL11A, a known silencer of the HBG1/2 genes, and in part by repressing the HBG1/2 genes via two direct binding sites. NFI factors emerge as versatile regulators of the fetal-to-adult switch in b-globin production.
2022-03-23 | GSE180855 | GEO
Project description:BCL11A represses gamma globin expression by binding to the gamma globin gene (HBG1 and HBG2) promoters. Genome editing of the BCL11A erythroid enhancer in the intron 2 of BCL11A gene or the BCL11A binding site at the HBG1/2 promoters disrupts this pathway and leads to gamma globin induction. Transcriptomic profiling of erythroid cells derived from human CD34+ cells edited at either target site using CRISPR-Cas9 revealed broader transcriptome perturbation when edited at the BCL11A erythroid enhancer.
2024-12-10 | GSE264491 | GEO
Project description:The mechanisms by which the fetal type b-globin-like genes HBG1 and HBG2 are silenced in adult erythroid precursor cells is a basic biology question in human development. Reversal of such mechanisms is beneficial for b hemoglobinopathies, such as sickle cell disease (SCD). A CRISPR-Cas9 genetic screen uncovered two members of the NFI transcription factor family – NFIA and NFIX – as novel HBG1/2 repressors. Both factors are expressed at elevated levels in adult erythroid cells, and their single or combined depletion revealed cooperativity in HBG1/2 regulation in cultured cells and human-to-mouse xenotransplant experiments, as well as in preventing sickling of SCD-derived erythroblasts. Genomic profiling, gene editing and in vitro binding assays demonstrated that the potent concerted activity of NFIA and NFIX factors is explained in part by their ability to activate expression of BCL11A, a known silencer of the HBG1/2 genes, and in part by repressing the HBG1/2 genes via two direct binding sites. NFI factors emerge as versatile regulators of the fetal-to-adult switch in b-globin production.
2022-03-23 | GSE180869 | GEO
Project description:The BAF (Brg/Brahma-associated factors) complex, also referred to as the mammalian Switch/Sucrose-Nonfermentable (mSWI/SNF) chromatin remodeling complex, plays a pivotal role in epigenetically regulating diverse transcriptional programs. BAF’s chromatin remodeling activity, which enhances accessibility to transcriptional machinery, is critical for gene regulation. We hypothesize that redirecting endogenous BAF complexes to “turn on” therapeutically relevant genes offer an additional approach for disease treatment. Using chronic myelogenous leukemia K562 cells stably expressing the FIRE-dCas9 system (K562-FIRE) and rapamycin as a chemical inducer of proximity (CIP) via CRISPR/Cas9 and FKBP/FRB dimerization, we redirected endogenous BAF complexes to the promoter of fetal hemoglobin (HBG) using pooled small guide RNAs (sgRNAs) targeting the HBG1 promoter. RNA-seq analysis showed a statistically significant increase in HBG1 and HBG2 gene expression in rapamycin treated cells over the DMSO treated controls.
2025-12-04 | GSE249747 | GEO
Project description:Despite extensive investigation, mechanisms of developmental hemoglobin expression remain incompletely understood. Hemoglobin switching is controlled by transcription factors, miRNAs, and RNA-binding proteins (RBPs) that enforce gene regulatory changes through development. Here we examine the role of the heterochronically silenced N-6 methyladenosine (m6A) RNA-binding protein, IGF2BP1 that was previously described to regulate HBG1/2 indirectly by suppressing BCL11A expression through a poorly understood mechanism. We find that IGF2BP1 binds and activates HIC2, itself a BCL11A repressor. Furthermore, we identify that IGF2BP1 plays a BCL11A-independent role by direct binding to HBG1/2 to promote its translation. Stop codon-proximal m6A-modified coding sequences within HBG2 transcripts are necessary and sufficient for direct positive regulation mediated by IGF2BP1. Together this work deepens the mechanistic understanding of hemoglobin switching and suggests a physical relationship between heterochronic RBPs and globin transcripts.
2025-08-04 | GSE303945 | GEO
Project description:Reactivation of fetal hemoglobin expression by down-regulation of BCL11A is a promising treatment of -hemoglobinopathies. A detailed understanding of BCL11A-mediated repression of -globin gene (HBG1/2) transcription is lacking, as studies to date used perturbations by shRNA or CRISPR/Cas9 gene editing. We leveraged the dTAG PROTAC platform to acutely deplete BCL11A protein in erythroid cells and examined consequences by PRO-seq, proteomics, chromatin accessibility, and histone profiling. Among ≤ 31 genes repressed by BCL11A, HBG1/2 and HBZ show the most abundant and progressive changes in transcription and chromatin accessibility upon BCL11A loss. Transcriptional changes at HBG1/2 were detected in < 2h. Robust HBG1/2 reactivation upon acute BCL11A-depletion occurred without loss of promoter 5methylcytosine (5mC). Using targeted protein degradation, we establish a hierarchy of gene reactivation at BCL11A targets, in which nascent transcription is followed by increased chromatin accessibility, and both are uncoupled from promoter DNA methylation at the HBG1/2 loci.
2023-10-24 | PXD030307 | Pride
Project description:Reactivation of fetal hemoglobin expression by down-regulation of BCL11A is a promising treatment of -hemoglobinopathies. A detailed understanding of BCL11A-mediated repression of -globin gene (HBG1/2) transcription is lacking, as studies to date used perturbations by shRNA or CRISPR/Cas9 gene editing. We leveraged the dTAG PROTAC platform to acutely deplete BCL11A protein in erythroid cells and examined consequences by PRO-seq, proteomics, chromatin accessibility, and histone profiling. Among ≤ 31 genes repressed by BCL11A, HBG1/2 and HBZ show the most abundant and progressive changes in transcription and chromatin accessibility upon BCL11A loss. Transcriptional changes at HBG1/2 were detected in < 2h. Robust HBG1/2 reactivation upon acute BCL11A-depletion occurred without loss of promoter 5methylcytosine (5mC). Using targeted protein degradation, we establish a hierarchy of gene reactivation at BCL11A targets, in which nascent transcription is followed by increased chromatin accessibility, and both are uncoupled from promoter DNA methylation at the HBG1/2 loci
2022-07-27 | PXD030301 | Pride
Project description:Multiple enhancers can coordinately regulate a target gene to ensure robust expression. Under the failure rate model, each redundant enhancer contributes a probability of target gene expression in the cell, and genetic deletion will cause single-cell expression phenotypes. However, this model has been challenging to test in human cells. To examine the role of the human β-globin enhancers in controlling single-cell expression phenotypes, we engineered a haploid locus in K562 cells that fluorescently reports the expression of HBG1. We isolated combinatorial enhancer deletion clones having single-cell expression phenotypes. Genetic and epigenetic analysis shows that deletion of HS3 or HS4 yields clonal cells having bimodal expression of HBG1. Time course analysis indicates that clonal cells can transition between bimodal expression states, and that GFP+ cells have more open chromatin at the HBG1 promoter than GFP- cells. Finally, we derive failure rate contributions for several enhancers. Thus, β-globin enhancers regulate bistable gene expression states, supporting the failure rate model of enhancer redundancy in human cells.
2022-01-21 | GSE192841 | GEO