Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA387385 | ENA |
REPOSITORIES: ENA
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Project description:Fetal and adult β-globin gene expression is tightly regulated during human development. Fetal globin genes are transcriptionally silenced during embryogenesis through the process of hemoglobin switching. Efforts to understand the transcriptional mechanism(s) behind fetal globin silencing have led to novel strategies to derepress fetal globin expression in the adult, which could alleviate symptoms in hereditary b-globin disorders including sickle cell disease (SCD) and β-thalassemia. We identified a novel zinc finger protein, pogo transposable element with zinc finger domain (Pogz), expressed in mouse and human hematopoietic stem and progenitor cells, which represses embryonic b-like globin gene expression in mice. Ablation of Pogz expression in adult hematopoietic cells in vivo results in persistence of embryonic b-like globin expression without significantly affecting erythroid development or mouse survival. Elevated embryonic β-like globin expression correlates with reduced expression of Bcl11a, a known repressor of embryonic β-like globin expression, in Pogz-/- fetal liver cells. Pogz binds to the Bcl11a promoter, and, to erythroid specific intragenic regulatory regions. Importantly, Pogz+/- mice develop normally, but show elevated embryonic b-like globin expression in peripheral blood cells, demonstrating that reducing Pogz levels results in persistence of embryonic b-like globin expression. Finally, knockdown of POGZ in primary human CD34+ hematopoietic stem and progenitor cell derived erythroblasts, reduces BCL11A expression and increases fetal hemoglobin expression. These findings are significant since new therapeutic targets and strategies are needed to treat the increasing global burden of b-globin disorders.
2018-06-21 | GSE113503 | GEO
Project description:RNA-seq of Ro60-null GM12878 cell lines in order to determine the gene expression changes resulting from loss of Ro60. 3 separate clones of Ro60(Trove2)-null cells derived from zinc finger nuclease targeting of exon 2, two wildtype biological replicates, +/- IFNa for 6 hours.
2015-09-16 | E-GEOD-72501 | biostudies-arrayexpress
Project description:Zinc finger nuclease knockouts of human ADP-glucokinase
2015-09-29 | E-GEOD-39497 | ExpressionAtlas
Project description:We have generated isogenic induced pluripotent stem cell lines by reprogramming human fibroblasts from patients carrying the LRRK2 G2019S mutation with subsequent zinc finger nuclease - mediated targeted correction of the diseased allele. These iPS cell lines were differentiated for 30 days using a direct differentiation protocol towards midbrain dopaminergic neurons (mDANs). Isogenic human iPS cells carrying the LRRK2 WT and G2019S locus were differentiated to dopaminergic neurons to detect gene expression changes associated with mutated LRRK2.
2013-03-07 | E-GEOD-43364 | biostudies-arrayexpress
Project description:Zinc finger nuclease knockouts of human ADP-glucokinase
| PRJNA170990 | ENA
Project description:Zinc Finger Nucleases (ZFNs) facilitate precise editing of DNA enabling targeted genomic modifications in vivo. ZFNs have been employed to obtain genetically modified plants and animals, and cell-based therapies utilizing ZFNs are undergoing clinical trials. However, many ZFNs display dose-dependent toxicity presumably due to the generation of undesired double stranded breaks at off-target sites within the genome. To evaluate the parameters influencing the functional specificity of ZFNs, we compared the in vivo activity of ZFN variants targeting the zebrafish kdrl locus, which display both high on-target activity and dose-dependent toxicity. We evaluated their functional specificity by assessing lesion frequency at 141 potential off-target sites within the zebrafish genome using Illumina sequencing. Only a minority of these off-target sites displayed significant lesion frequency with kdrl ZFNs. Furthermore, we find that active off-target sites appear to be defined by the thermodynamics of zinc finger-DNA recognition. Surprisingly, we observed that the zinc finger protein specificity and the choice of the engineered dimerization domain of the FokI nuclease could independently influence the fidelity of these ZFNs. The results of this study have implications for the assessment of likely off-target sites within a genome and point to both ZFP-dependent and –independent mechanisms of potential improvement for engineering ZFNs with higher levels of precision. Examined lesions at 141 off-target sites for various treatments of ZFNs and compare to the untreated sample stage 1: raw read but missing quality values stage 2: fastq files available from SRA
2010-08-25 | E-GEOD-23762 | biostudies-arrayexpress
Project description:Fetal hemoglobin (HbF) level is genetically controlled and modifies severity of adult hemoglobin (HbA) disorders. Common genetic variation affects expression of BCL11A, a critical regulator of HbF silencing. Current models suggest that BCL11A acts at a distance from the gamma-globin genes via long-distance chromosomal interactions. Here we use a functional cellular assay and protein-binding microarray to establish a requirement for a zinc-finger cluster of BCL11A for globin repression, and identify a preferred DNA recognition sequence (TGACCA). The motif is present in embryonic and fetal-expressed globin promoters, and duplicated in gamma-globin promoters, yet only the distal motif is mutated in alleles of individuals with hereditary persistence of hemoglobin. Using CUT&RUN to map protein binding sites, we detected BCL11A occupancy preferentially at the distal motif, and validated its absence in HbF-expressing, promoter-edited erythroid cells. Taken together, our findings reveal that direct gamma-globin gene promoter repression by BCL11A underlies hemoglobin switching.
2018-03-30 | GSE104676 | GEO
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:The goal of this study was to characterize a novel Mecp2 allele in the laboratory rat, a distinct rodent species from the laboratory mouse with unique features. The allele was created by zinc finger-nuclease (ZFN) targeting (SAGE/Horizon) of the X-linked gene, Methyl-CpG-Binding Protein 2 (Mecp2), resulting in normal Mecp2 RNA abundance, but absent protein in male rats as expected due to the presence of only one copy of mutant Mecp2, and an approximate 50% reduction in female rats as expected from animals that harbor one wild-type copy and one mutant copy of Mecp2. Behavioral characterization of female rats with the Mecp2 ZFN allele and wild-type littermates was conducted, and Mecp2 ZFN/+ female rats showed behavioral phenotypes that were consistent with disease-like features present during the early stages of disease onset in the neurological disorder Rett syndrome. The goal of conducting RNAseq studies was to compare existing gene expression alterations in the Mecp2 rat with one of the most widely studied Mecp2 mouse model. Hypothalami were obtained from males with complete loss of MeCP2 (ZFN/y) and wild-type littermate male rats for RNA-seq studies. Common and unique gene expression alterations among the Mecp2 rodents that were then tested in human Rett and control postmortem brain revealed the benefit of combining findings from both models, suggesting the predictive validity of this approach for future studies for the identification of potential preclinical outcome measures. QPCR validation in an independent set of rats was conducted with a subset of genes from the Mecp2 ZFN rat RNA-seq data as an additional control measure. Taken together, these findings demonstrate that the Mecp2 rat model is a complementary tool with unique features for the study of RTT and highlight the potential benefit of cross-species analyses in identifying potential disease-relevant preclinical outcome measures. Transcriptional profiles of hypothalamic samples obtained from male rats haboring a novel zinc-finger nuclease Mecp2 loss-of-function allele and corresponding wild-type littermate rats were generated using RNA-seq
2016-08-09 | E-GEOD-83323 | biostudies-arrayexpress
Project description:Zinc Finger Nucleases (ZFNs) facilitate precise editing of DNA enabling targeted genomic modifications in vivo. ZFNs have been employed to obtain genetically modified plants and animals, and cell-based therapies utilizing ZFNs are undergoing clinical trials. However, many ZFNs display dose-dependent toxicity presumably due to the generation of undesired double stranded breaks at off-target sites within the genome. To evaluate the parameters influencing the functional specificity of ZFNs, we compared the in vivo activity of ZFN variants targeting the zebrafish kdrl locus, which display both high on-target activity and dose-dependent toxicity. We evaluated their functional specificity by assessing lesion frequency at 141 potential off-target sites within the zebrafish genome using Illumina sequencing. Only a minority of these off-target sites displayed significant lesion frequency with kdrl ZFNs. Furthermore, we find that active off-target sites appear to be defined by the thermodynamics of zinc finger-DNA recognition. Surprisingly, we observed that the zinc finger protein specificity and the choice of the engineered dimerization domain of the FokI nuclease could independently influence the fidelity of these ZFNs. The results of this study have implications for the assessment of likely off-target sites within a genome and point to both ZFP-dependent and –independent mechanisms of potential improvement for engineering ZFNs with higher levels of precision.
2010-08-25 | GSE23762 | GEO