MiRNA expression analysis of erythroblasts derived from SCD CD34+ HSPCs transduced with the βAS3m/miR7m compared to control- (βAS3m/miRnt or βAS3) or mock-transduced samples
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE205073 | GEO | 2023/05/24
REPOSITORIES: GEO
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Project description:We have investigated gene expression changes occurring in samples transduced with a lentiviral vector expressing the βAS3m transgene and the miR7m targeting the sickle β-globin
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Project description:The molecular mechanisms underlying erythroid-specific gene regulation remain incompletely understood. Closely spaced binding sites for GATA, NF-E2/maf and CACCC interacting transcription factors play functionally important roles in globin and other erythroid-specific gene expression. We and others recently identified the CACCC-binding transcription factor ZBP-89 as a novel GATA-1 and NF-E2/mafK interacting partner. Here, we examined the role of ZBP-89 in human globin gene regulation and erythroid maturation using a primary CD34+ cell ex vivo differentiation system. We show that ZBP-89 protein levels rise dramatically during human erythroid differentiation, and that ZBP-89 occupies key cis-regulatory elements within the globin and other erythroid gene loci. ZBP-89 binding correlates strongly with RNA Pol II occupancy, active histone marks, and high-level gene expression. ZBP-89 physically associates with the histone acetyltransferases (HATs) p300 and Gcn5/Trrap, and occupies common sites with Gcn5 within the human globin loci. Lentiviral shRNA knockdown of ZBP-89 results in reduced Gcn5 occupancy, decreased acetylated histone 3 levels, lower globin and erythroid-specific gene expression, and impaired erythroid maturation. Addition of the HDAC inhibitor valproic acid partially reverses the reduced globin gene expression. These findings reveal an activating role for ZBP-89 in human globin gene regulation and erythroid differentiation. Keywords: Antibody ChIP-chip, Human Primary Erythroblasts
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Project description:Room temperature whole blood mRNA stabilization procedures, such as the PAX gene system, are critical for the application of transcriptional analysis to population-based clinical studies. Global transcriptome analysis of whole blood RNA using microarrays has proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA in the blood. This is a particular problem in patients with sickle-cell disease, secondary to the high abundance of globin-expressing nucleated red blood cells and reticulocytes in the circulation . In order to more accurately measure the steady state whole blood transcriptome in sickle-cell patients, we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA samples for genome-wide transcriptome analyses using oligonucleotide arrays. We demonstrate here by both microarrays and Q-PCR that the globin mRNA depletion method resulted in 55-65 fold reduction in globin transcripts in whole blood collected from healthy volunteers and sickle-cell disease patients. This led to an improvement in microarray data quality with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. The differentially modulated genes from the globin depleted samples had a higher correlation coefficient to the 112 genes identified to be significantly altered in our previous study on sickle-cell disease using PBMC preparations. Additionally, the analysis of differences between the whole blood transcriptome and PBMC transcriptome reveals important erythrocyte genes that participate in sickle-cell pathogenesis and compensation. The combination of globin mRNA reduction after whole-blood RNA stabilization represents a robust clinical research methodology for the discovery of biomarkers for hematologic diseases and in multicenter clinical trials investigating a wide range of nonhematologic disorders where fractionation of cell types is impracticable. Keywords: Microarrays, PAXgene, globin reduction, whole blood, PBMC There are 10 samples for each of PBMC, PAX and PAX globin-reduced, where 5 samples come from sickle-cell patients and 5 from healthy controls.
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Project description:Robust β-globin expression in erythroid cells derived from induced pluripotent stem cells (iPSCs) would increase the resolution with which red blood cell disorders such as sickle cell disease and β thalassemia can be modeled in vitro. To better quantify efforts in augmenting β-globin expression, we report the creation of a β-globin reporter iPSC line through the insertion of a GFP cassette after the endogenous β-globin promoter, allowing for the mapping of β-globin expression throughout erythroid development in real time at single cell resolution. Sorting live GFP+ and GFP- cells at the most mature stage of erythroid differentiation, followed by single cell RNA sequencing (scRNAseq), identifies features that distinguish GFP- from GFP+ β-globin expressing cells and allows for the dissection of the developmental and maturational status of iPSC-derived erythroid cells. Co-expression of embryonic, fetal and adult globins in individual cells indicates a yolk sac erythro-myeloid progenitor (EMP) stage of hematopoietic development, representing the onset of definitive erythropoiesis. Within this developmental program, scRNAseq analysis identifies a gradient of erythroid maturation with GFP+ β-globin expressing cells showing increased maturation. In addition, scRNAseq analysis reveals that definitively patterned iPSC-derived erythroblasts resemble their postnatal counterparts in terms of gene expression and essential biological processes, confirming their potential for disease modeling and regenerative medicine applications.
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Project description:Reactivation of gamma-globin is considered a promising approach for the treatment of beta-thalassaemia and sickle cell disease. Therapeutic induction of gamma-globin expression is fraught with lack of suitable therapeutic targets. In order to identify new potential targets we analysed the changes in the proteome of human primary erythroid progenitor cells by treatment with decitabine, a known, yet not clinically safe, gamma-globin inducer. Significant differentially expressed proteins were identified which were involved in various biological pathways and functional categories.
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Project description:Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the beta-thalassemia syndromes. Genetic association studies have identified sequence variants in the gene BCL11A that influence HbF levels. Here we examine BCL11A as a potential regulator of HbF expression. The high HbF BCL11A genotype is associated with reduced BCL11A expression. Moreover, abundant expression of full-length forms of BCL11A is developmentally restricted to adult erythroid cells. Down-regulation of BCL11A expression in primary adult erythroid cells leads to robust HbF expression. Consistent with a direct role of BCL11A in globin gene regulation, we find that BCL11A occupies several discrete sites in the beta-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in beta-hemoglobin disorders. BCL11A siRNA label: B, NT siRNA label: N Keywords: cell type comparsion
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Project description:Human fetal γ-globin gene is developmentally silenced around the birth, and reactivation of γ-globin gene in adulthood sheds new light on ameliorating symptoms of hemoglobin disorders, such as sickle cell disease (SCD) and β-thalassemias. However, the precise regulation process of γ-globin remains incompletely understood. Here, we found that a new protein directly interacted with SOX6 and exerted a significant repression effect on the expression of γ-globin gene in erythroid cells. Further studies have demonstrated that it bound directly to γ-globin gene promoter via octamer binding motif, which in turn suppressed the transcriptional activity of γ-globin gene promoter. Thus, these data indicate that this new protein acts as a novel transcriptional repressor in the regulation of γ-globin gene expression through direct promoter binding, implying a potential alternative therapeutic target for the treatment of SCD and β-thalassemias.
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Project description:Room temperature whole blood mRNA stabilization procedures, such as the PAX gene system, are critical for the application of transcriptional analysis to population-based clinical studies. Global transcriptome analysis of whole blood RNA using microarrays has proven to be challenging due to the high abundance of globin transcripts that constitute 70% of whole blood mRNA in the blood. This is a particular problem in patients with sickle-cell disease, secondary to the high abundance of globin-expressing nucleated red blood cells and reticulocytes in the circulation . In order to more accurately measure the steady state whole blood transcriptome in sickle-cell patients, we evaluated the efficacy of reducing globin transcripts in PAXgene stabilized RNA samples for genome-wide transcriptome analyses using oligonucleotide arrays. We demonstrate here by both microarrays and Q-PCR that the globin mRNA depletion method resulted in 55-65 fold reduction in globin transcripts in whole blood collected from healthy volunteers and sickle-cell disease patients. This led to an improvement in microarray data quality with increased detection rate of expressed genes and improved overlap with the expression signatures of isolated peripheral blood mononuclear (PBMC) preparations. The differentially modulated genes from the globin depleted samples had a higher correlation coefficient to the 112 genes identified to be significantly altered in our previous study on sickle-cell disease using PBMC preparations. Additionally, the analysis of differences between the whole blood transcriptome and PBMC transcriptome reveals important erythrocyte genes that participate in sickle-cell pathogenesis and compensation. The combination of globin mRNA reduction after whole-blood RNA stabilization represents a robust clinical research methodology for the discovery of biomarkers for hematologic diseases and in multicenter clinical trials investigating a wide range of nonhematologic disorders where fractionation of cell types is impracticable. Keywords: Microarrays, PAXgene, globin reduction, whole blood, PBMC
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