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Analysis of nascent transcription by PRO-seq in HUDEP-2 cells following CRISPR editing of BCL11A enhancer

ABSTRACT: CRISPR editing of an intronic enhancer within the BCL11A gene reactivates fetal hemoglobin (HbF) in adult erythroid cells, serving as a gene-based therapy for hemoglobinopathies. However, the mechanisms underlying the remarkable efficacy of CRISPR-mediated enhancer ablation remain poorly understood. Ehancer RNAs transcribes from enhancer play a role in gene regulation. Here, we apply PRO-seq in wild-type and sg1617-edited HUDEP-2 cells to determine the effects of sg1617 editing on enhancer RNA transcription.

ORGANISM(S): Homo sapiens

PROVIDER: GSE292697 | GEO | 2025/11/13

REPOSITORIES: GEO

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Analysis of nascent transcription by PRO-cap in HUDEP-2 cells

Project description:CRISPR editing of an intronic enhancer within the BCL11A gene reactivates fetal hemoglobin (HbF) in adult erythroid cells, offering a potential gene-based therapy for hemoglobinopathies. However, the mechanisms underlying the remarkable efficacy of CRISPR-mediated enhancer ablation remain poorly understood. Enhancer RNAs, transcribed from enhancers, play a role in gene regulation. We apply PRO-cap to map the location of active polymerases and transcription start sites.

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Analysis of chromatin structure of the BCL11A locus in HUDEP-2 cells by Capture-Pore-C (CPC)

Project description:CRISPR editing of an intronic enhancer within the BCL11A gene reactivates fetal hemoglobin (HbF) in adult erythroid cells, serving as a gene-based therapy for hemoglobinopathies. However, the mechanisms underlying the remarkable efficacy of CRISPR-mediated enhancer ablation remain poorly understood. We describe an evolutionarily conserved, enhancer-dependent chromatin architecture, the enhancer-CTCF rosette, essential for chromatin insulation and the developmental expression of BCL11A in hematopoiesis. CRISPR-mediated disruption of lineage-specific BCL11A enhancers impairs enhancer RNA transcription and NIPBL-cohesin loading, leading to destabilized chromatin configuration, impaired chromatin insulation, and epigenetic silencing of BCL11A. Antisense oligonucleotide-mediated depletion of enhancer RNA silences BCL11A by disrupting chromatin insulation in adult erythroid cells.

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Analysis of chromatin accessbility in erythroid cells following CRISPR editing or ASO perturbation of BCL11A enhancer

2025-11-13 | GSE292669 | GEO

Global transcriptions upon MAZ knockdown HUDEP-2 cells

Project description:Hemoglobinopathies, such as sickle cell disease and β-thalassemia, are common genetic disorders remaining significant global health challenges due to their associated morbidity and mortality. Increasing fetal hemoglobin (HbF) levels has emerged as a promising therapeutic strategy for these disorders. In this study, we report MAZ as a repressor of γ-globin expression in human erythroid cells. Depletion of MAZ in HUDEP-2 and patient-derived β-thalassemia cells leads to significant inductions both of γ-globin mRNA and protein levels, resulting in increased HbF percentages and HbF+ erythroid cells. We demonstrate that MAZ occupies at the MYB promoter. MAZ depletion reduced MYB and BCL11A levels. Restoration of MYB re-silenced the γ-globin levels in MAZ depleted cells. Our findings uncover the MAZ-MYB axis in γ-globin regulation, highlighting MAZ as a potential target to enhance HbF levels in patients with hemoglobin disorders.

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Multiplex base editing of BCL11A regulatory elements to treat sickle cell disease

Project description:Sickle cell disease (SCD) is a genetic anemia caused by the production of an abnormal adult hemoglobin. The clinical severity is lessened by elevated fetal hemoglobin (HbF) production in adulthood. A promising therapy is the transplantation of autologous, hematopoietic stem/progenitor cells (HSPCs) treated with CRISPR/Cas9 to downregulate the HbF repressor BCL11A via generation of double strand breaks (DSBs) in the +58-kb erythroid-specific enhancer. Here, to further enhance HbF production without increasing the mutagenic load, we targeted both +58-kb and +55-kb BCL11A erythroid-specific enhancers using base editors. We systematically dissected DNA motifs recognized by the key transcriptional activators within these regions and identified the critical nucleotides required for activator binding. Multiplex base editing of these residues was efficient and safe and generated no or little DSBs and genomic rearrangements. We observed substantial HbF reactivation, exceeding the levels achieved using the CRISPR/Cas9 nuclease-based strategy, thus efficiently rescuing the sickling phenotype. Multiplex base editing was efficient in long-term repopulating HSPCs and resulted in potent HbF reactivation in vivo. In summary, these results show that multiplex base editing of BCL11A erythroid-specific enhancers is a safe and potent strategy for treating sickle cell disease.

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Micro-Capture-C to determine the long-range interaction of HBG1/2 promoter with the locus control region (LCR), a powerful upstream enhancer in -175 A>G mutantant HUDEP-2 cells.

Project description:Naturally occurring mutations in the γ-globin promoters can result in hereditary persistence of fetal hemoglobin (HPFH), a benign condition that ameliorates the severity of β-hemoglobinopathies through increased post-natal fetal hemoglobin (HbF) expression. Base editing to recreate the HPFH mutations is a promising approach to induce HbF. Compared with Cas9-generated indels, base-editing approaches were more potent, with the −175 A>G conversion resulting in the strongest induction of HbF by creating a new TAL1 binding motif that stimulates long-range interaction with the locus control region (LCR), a powerful upstream enhancer. Micro-Capture-C analysis showed that introducing the −175 A>G variant stimulated long-range interaction between the γ-globin promoters and the LCR in erythroid progenitor cell line. Together, these findings show that the −175 A>G variant creates a bipartite GATA–TAL1 motif, resulting in the assembly of a GATA1/TAL1/LMO2/LDB1 complex, which activates γ-globin gene transcription by enhancing its physical association with the LCR.

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Analysis of nascent transcription by PRO-seq in HUDEP-2 cells following CRISPR editing of BCL11A enhancer

Project description:Analysis of nascent transcription by PRO-seq in HUDEP-2 cells following CRISPR editing of BCL11A enhancer

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The LRF/ZBTB7A transcription factor is a BCL11A-independent repressor of fetal hemoglobin

Project description:Hemoglobinopathies, including sickle cell disease and _-thalassemia, are global public health concerns. Induction of fetal-type hemoglobin (HbF) is a promising means to treat these disorders; however, precisely how HbF expression is silenced in adult erythroid cells is not fully understood. Here, we show that the LRF/ZBTB7A transcription factor is a potent repressor of HbF production. LRF inactivation derepresses embryonic/fetal _-globin expression in mouse and human adult erythroid cells. We employed genome-wide analysis of the transcriptome, chromatin accessibility and LRF occupancy sites, and demonstrate that LRF occupies the _-globin loci and maintains nucleosome density necessary for _-globin silencing. LRF confers its repressive activity through a unique NuRD repressor complex independent of BCL11A. Strikingly, human erythroid lines lacking both LRF and BCL11A exhibited almost a complete switch in expression from adult- to fetal-type globin, suggesting that these two factors cumulatively represent the near entirety of _-globin repressive activity in adult erythroid cells. RNA-seq, LRF ChIP-seq and ATAC-seq assays were used to investigtae LRF binding, effect of LRF depletion on transcription and chromatin landscape in mouse and human cells.

2016-01-15 | E-GEOD-74977 | biostudies-arrayexpress

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