RNA-seq and CUT&RUN of erythroblasts upon perturbation of gamma-hemoglobin repressors
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE247096 | GEO | 2024/06/19
REPOSITORIES: GEO
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Project description:Through high throughput compound screening, we've identified compounds that induce the expression of fetal hemoglobin. This study contains CUT&RUN data of a novel target, WIZ.
2024-06-19 | E-MTAB-13890 | biostudies-arrayexpress
Project description:BCL11A, the major regulator of HbF(α2γ2) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult adult-type HbA (α2β2) production. Yet, the mechanism remains unclear. To uncover how BCL11A initiates repression, we used CRISPR/Cas9 and dCas9 screens to dissect the γ-globin promoters and identified an apparent activator element near the BCL11A binding region. Using CUT&RUN and base editing approaches, we demonstrate that this element, the proximal CCAAT box, is the binding site of transcription activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate γ-globin repression, and the distance between the two motifs is critical for direct competition. Occupancy of NF-Y is rapidly established upon BCL11A depletion, and precedes γ-globin derepression and LCR-globin loop formation. Our findings reveal that the critical fetal-to-adult hemoglobin switch is initiated by the competition between transcription factors within a discrete region in the γ-globin promoters.
2020-12-22 | GSE150529 | GEO
Project description:The fetal (HbF)-to-adult (HbA) hemoglobin switch is a paradigm for developmental gene expression control with relevance to sickle cell disease and beta-thalassemia. Polycomb repressive complex (PRC) proteins regulate this switch, and an inhibitor of the EED subunit of PRC2 entered clinical trials for fetal hemoglobin activation. Yet, how PcG complexes function in this process, their target genes and relevant subunit composition are unknown. Here, we identified the PRC1 subunit BMI1 as a novel HbF repressor. We uncovered LIN28B, IGF2BP1, and IGF2BP3 as direct BMI1 targets and demonstrate that they account for the BMI1 effects. BMI1 functions as part of the canonical PRC1 (cPRC1) subcomplex as revealed by the physical and functional dissection of BMI1 protein partners. Lastly, we demonstrate that BMI1/cPRC1 acts in concert with the PRC2 to repress HbF through the same target genes. Our study illuminates how PRC silences HbF, highlighting an epigenetic mechanism involved in hemoglobin switching.
2023-03-01 | GSE218231 | GEO
Project description:Transcription factor competition at the γ-globin promoters controls hemoglobin switching [CUT&RUN]
| PRJNA632652 | ENA
Project description:The switch from fetal to adult hemoglobin production has been studied in great depth in part because of its relevance to the treatment of hemolobinopathies. Transcription factor BCL11A, which is essential for repression of the fetal beta-type globin (γ-globin) genes after birth, is largely controlled at the level of transcription but the mechanism of BCL11A developmental control is unknown. Here, using a CRISPR-Cas9 screen in human erythroblasts, we identify transcription factor HIC2 as a repressor of BCL11A transcription. HIC2 and BCL11A expression are anti-correlated in fetal and adult erythroblasts. Forced expression of HIC2 in adult erythroblasts silences BCL11A transcription and markedly induces γ-globin expression. HIC2 binds selectively to constituent erythroid developmental BCL11A enhancer to reduce chromatin accessibility and impair access by transcription factor GATA1, resulting in loss of enhancer activity and enhancer-promoter contacts. Conversely, loss of HIC2 in fetal erythroblasts increases enhancer accessibility, enables GATA1 binding and induces BCL11A transcription. HIC2 is unveiled as a critical evolutionarily conserved regulator of globin gene switching by imposing developmental control on the BCL11A gene.
2022-05-26 | GSE182530 | GEO
Project description:NFI factors repress fetal hemoglobin in adult erythorid cells [CUT&RUN]
| PRJNA749785 | ENA
Project description:Genetic manipulations to increase fetal hemoglobin (HbF, α2γ2) in postnatal red blood cells (RBCs) can alleviate β-thalassemia and sickle cell disease. We compared five strategies in CD34+ hematopoietic stem and progenitor cells, using either Cas9 nuclease, which creates uncontrolled indel mixtures, or adenine base editors (ABE), which generate more precise nucleotide changes. The most potent modification was ABE generation of γ-globin (HBG1/HBG2) −175 A>G, which creates a promoter binding motif for the transcriptional activator TAL1. In erythroid colonies with >87.5% on-target edits, those with −175 A>G expressed 81 ± 7% HbF, versus 17 ± 11% in unedited controls. In comparison, HbF levels were lower and more variable in erythroid cells modified with either of two Cas9 nuclease strategies with similar editing efficiencies, being 32 ± 19% when a BCL11A repressor binding motif in the γ-globin promoter was targeted and 52 ± 13% when the +58 BCL11A erythroid enhancer was targeted. Contrary to currently accepted models of γ-globin regulation, HbF levels varied significantly with different Cas9 indels that disrupted the γ-globin promoter BCL11A binding motif. The −175 A>G base edit also induced HbF more potently than did the Cas9 nuclease approaches in RBCs generated after transplantation of modified normal or SCD patient CD34+ cells into mice. Our data suggest a strategy for potent, uniform induction of HbF and provide insights into γ-globin gene regulation. More generally, we demonstrate that diverse indels generated by Cas9 nuclease can cause unexpected variations in biological outcomes that can be circumvented by base editing, with important implications for therapeutic gene editing efforts.
2023-04-28 | GSE228819 | GEO
Project description:BACKGROUND & AIMS: Recent studies revealed that hemoglobin is expressed in some non-erythrocytes and it suppresses oxidative stress when overexpressed. Oxidative stress plays a critical role in the pathogenesis of non-alcoholic steatohepatitis (NASH). This study was to investigate whether hemoglobin is expressed in hepatocytes and how it is related to oxidative stress in NASH patients. METHODS: Microarray was performed to identify differentially expressed genes in NASH. Quantitative real time PCR (qRT-PCR) was used to examine gene expression levels. Western blotting and immunofluorescence staining were employed to examine hemoglobin proteins. Flow cytometry was used to measure intracellular oxidative stress. RESULTS: Analysis of microarray gene expression data has revealed a significant increase in the expression of hemoglobin alpha (HBA1) and beta (HBB) in liver biopspies from NASH patients. Increased hemoglobin expression in NASH was validated by qRT-PCR. However, the expression of erythrocyte specific marker genes such as SPTA, SPTB, GYPA, GATA1, and ALAS2 did not change, indicating that increased hemoglobin expression in NASH was not from erythropoiesis, but could result from increased expression in hepatocytes. Immunofluorescence staining demonstrated positive HBA1 and HBB expression in the hepatocytes of NASH livers. Hemoglobin expression was also observed in human hepatocellular carcinoma HepG2 cell line. Furthermore, treatment with hydrogen peroxide, a known oxidative stress inducer, induced a dose dependent increase in HBA1 expression in HepG2 cells. Intriguingly, forced hemoglobin expression suppressed oxidative stress. CONCLUSIONS: Oxidative stress upregulates hemoglobin expression in hepatocytes. Suppression of oxidative stress by hemoglobin could be a mechanism to protect hepatocytes from oxidative damage. These findings suggest that hemoglobin is an inducible antioxidant in hepatocytes in response to increased oxidative stress as found in NASH livers. Twelve biopsy diagnosed NASH patients were included in this study. For control groups, total RNA from 5 different subjects were purchased from ADMET. These subjects are free from liver disease.
2010-10-20 | E-GEOD-24807 | biostudies-arrayexpress
Project description:HIC2 represses BCL11A transcription to regulate hemoglobin switching during development [CUT&RUN]
| PRJNA756674 | ENA
Project description:BACKGROUND & AIMS: Recent studies revealed that hemoglobin is expressed in some non-erythrocytes and it suppresses oxidative stress when overexpressed. Oxidative stress plays a critical role in the pathogenesis of non-alcoholic steatohepatitis (NASH). This study was to investigate whether hemoglobin is expressed in hepatocytes and how it is related to oxidative stress in NASH patients. METHODS: Microarray was performed to identify differentially expressed genes in NASH. Quantitative real time PCR (qRT-PCR) was used to examine gene expression levels. Western blotting and immunofluorescence staining were employed to examine hemoglobin proteins. Flow cytometry was used to measure intracellular oxidative stress. RESULTS: Analysis of microarray gene expression data has revealed a significant increase in the expression of hemoglobin alpha (HBA1) and beta (HBB) in liver biopspies from NASH patients. Increased hemoglobin expression in NASH was validated by qRT-PCR. However, the expression of erythrocyte specific marker genes such as SPTA, SPTB, GYPA, GATA1, and ALAS2 did not change, indicating that increased hemoglobin expression in NASH was not from erythropoiesis, but could result from increased expression in hepatocytes. Immunofluorescence staining demonstrated positive HBA1 and HBB expression in the hepatocytes of NASH livers. Hemoglobin expression was also observed in human hepatocellular carcinoma HepG2 cell line. Furthermore, treatment with hydrogen peroxide, a known oxidative stress inducer, induced a dose dependent increase in HBA1 expression in HepG2 cells. Intriguingly, forced hemoglobin expression suppressed oxidative stress. CONCLUSIONS: Oxidative stress upregulates hemoglobin expression in hepatocytes. Suppression of oxidative stress by hemoglobin could be a mechanism to protect hepatocytes from oxidative damage. These findings suggest that hemoglobin is an inducible antioxidant in hepatocytes in response to increased oxidative stress as found in NASH livers.
2010-10-21 | GSE24807 | GEO