Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1152576 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR30401673_1.fastq.gz | Fastqsanger.gz | |||
| SRR30401673_2.fastq.gz | Fastqsanger.gz | |||
| SRR30401674_1.fastq.gz | Fastqsanger.gz | |||
| SRR30401674_2.fastq.gz | Fastqsanger.gz | |||
| SRR30401675_1.fastq.gz | Fastqsanger.gz |
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Project description:Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease resulting from loss-of-function mutations in SEC23B, which encodes a component of coat complex protein II (COPII) vesicles. We have previously shown that the expression of SEC23A, a paralogous protein for SEC23B, is significantly lower than that of SEC23B in human erythroid cells and that increased SEC23A expression rescues CDAII. In this study, we generated a human erythroid cell line that expresses eGFP from the endogenous genomic locus of SEC23A and performed a small molecule screen to identify compounds that increase the SEC23A-eGFP level. The top compound passing all filters was an LSD1 inhibitor. We validated that LSD1 inhibition with RN1 i) results in increased SEC23A mRNA and protein levels in primary erythroid cells derived from CD34+ human hematopoietic stem and progenitor cells (HSPCs), at doses that do not impair erythroid cell growth or differentiation and ii) rescues the defect resulting from SEC23B deletion in primary human erythroid cells. We validated LSD1 as the target of RN1 by showing that genetic downregulation of LSD1 also led to a profound induction of SEC23A mRNA levels in HPSC-derived erythroid cells. Subsequently, we validated our findings in vivo, demonstrating that deletion of Lsd1 in mouse erythroid cells results in increased Sec23a expression and that RN1 treatment rescues the erythroid defect observed in CDAII mice. Finally, using CUT&RUN, we found that LSD1 occupies a sequence in the SEC23A promoter, which when deleted, results in increased SEC23A expression and rescue of CDAII. These finding suggest that LSD1 occupies the SEC23A promoter, repressing SEC23A transcription and that in the setting of LSD1 inhibition, SEC23A expression is de-repressed, resulting in CDAII rescue. Therefore, using an unbiased screen, we have identified and validated a novel promising therapeutic strategy for CDAII, using a compound that inhibit LSD1 or a genetic approach based on deleting the SEC23A promoter sequence that is occupied by LSD1.
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Project description:Congenital dyserythropoietic anemia Type 1 (CDA1) is a rare macrocytic anemia caused by loss-of-function mutation of CDAN1. To investigate the functional role of CDAN1, we performed RNA-sequencing on human erythroleukemic K562 cells with CDAN1 shRNA knockdown using a doxycycline inducible system
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Project description:Analysis of erythroblastic cells differentiated from induced pluripotent stem cells (iPSCs) generated from peripheral blood T lymphocytes of CDA type IV patient. Type IV congenital dyserythropoietic anemia (CDA) is due to a monoallelic mutation at the second zinc finger of KLF1 (c.973G>A). Results provide insight into molecular mechanisms underlying CDA pathogenesis.
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Project description:Genetic disarray follows mutant KLF1-E325K expression in a congenital dyserythropoietic anemia patient
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Project description:Genome Wides Association Studies (GWAS) have identified tens of thousands of associations between human genetic variation and common disease. Despite the abundance of GWAS associations, functional identification and characterization of causative variants and effector genes remains a challenging prospect. Human erythropoiesis provides a highly tractable model system for the development of tools for GWAS analysis. We have developed a three-phase protocol for differentiation of peripheral CD34+ Haematopoietic Stem Cells (HSC) into mature enucleating erythrocytes. This protocol has been characterized for it’s the changes observed morphologically, immunologically (by FACS) and epigenetically (by ATAC-seq, ChIP-seq, and RNA-seq). We have used this protocol to study the effects of common GWAS variants affecting red blood cell traits, and more severe mutations causing Type-1 Congenital Dyserythropoietic Anemia (CDA-I).
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Project description:Genome Wides Association Studies (GWAS) have identified tens of thousands of associations between human genetic variation and common disease. Despite the abundance of GWAS associations, functional identification and characterization of causative variants and effector genes remains a challenging prospect. Human erythropoiesis provides a highly tractable model system for the development of tools for GWAS analysis. We have developed a three-phase protocol for differentiation of peripheral CD34+ Haematopoietic Stem Cells (HSC) into mature enucleating erythrocytes. This protocol has been characterized for it’s the changes observed morphologically, immunologically (by FACS) and epigenetically (by ATAC-seq, ChIP-seq, and RNA-seq). We have used this protocol to study the effects of common GWAS variants affecting red blood cell traits, and more severe mutations causing Type-1 Congenital Dyserythropoietic Anemia (CDA-I).
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Project description:Genome Wides Association Studies (GWAS) have identified tens of thousands of associations between human genetic variation and common disease. Despite the abundance of GWAS associations, functional identification and characterization of causative variants and effector genes remains a challenging prospect. Human erythropoiesis provides a highly tractable model system for the development of tools for GWAS analysis. We have developed a three-phase protocol for differentiation of peripheral CD34+ Haematopoietic Stem Cells (HSC) into mature enucleating erythrocytes. This protocol has been characterized for it’s the changes observed morphologically, immunologically (by FACS) and epigenetically (by ATAC-seq, ChIP-seq, and RNA-seq). We have used this protocol to study the effects of common GWAS variants affecting red blood cell traits, and more severe mutations causing Type-1 Congenital Dyserythropoietic Anemia (CDA-I).
2023-01-21 | GSE125925 | GEO
Project description:Genome Wides Association Studies (GWAS) have identified tens of thousands of associations between human genetic variation and common disease. Despite the abundance of GWAS associations, functional identification and characterization of causative variants and effector genes remains a challenging prospect. Human erythropoiesis provides a highly tractable model system for the development of tools for GWAS analysis. We have developed a three-phase protocol for differentiation of peripheral CD34+ Haematopoietic Stem Cells (HSC) into mature enucleating erythrocytes. This protocol has been characterized for it’s the changes observed morphologically, immunologically (by FACS) and epigenetically (by ATAC-seq, ChIP-seq, and RNA-seq). We have used this protocol to study the effects of common GWAS variants affecting red blood cell traits, and more severe mutations causing Type-1 Congenital Dyserythropoietic Anemia (CDA-I).
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Project description:Congenital dyserythropoietic anemia (CDA) type IV is caused by a heterozygous mutation, Glu325Lys (E325K), in the KLF1 transcription factor. A molecular understanding of this disease is absent, partly due to its rarity. We expanded erythroid cells from a patient’s peripheral blood and analyzed its global expression pattern. We find that a large number of erythroid pathways are disrupted, particularly those related to membrane transport, globin regulation, and iron utilization. The altered genetics lead to significant deficits in differentiation. Glu325 is within the KLF1 zinc finger domain at an amino acid critical for site specific DNA binding. The change to Lys is predicted to significantly alter the target site recognition sequence, both by subverting normal recognition and by enabling interaction with novel sites. Consistent with this, we find high level ectopic expression of genes not normally present in the red cell. These altered properties explain the patients’ clinical and phenotypic features and elucidate the dominant character of the mutation.
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