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Primary human CD34+ cells for modeling ETO2-GLIS2 leukemogenesis - Single cell RNAseq

ABSTRACT: Primary human CD34+ cells for modeling ETO2-GLIS2 leukemogenesis - Single cell RNAseq

PROVIDER: PRJEB76272 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR13189369_1.fastq.gz	Fastqsanger.gz
	ERR13189369_2.fastq.gz	Fastqsanger.gz
	ERR13189370_1.fastq.gz	Fastqsanger.gz
	ERR13189370_2.fastq.gz	Fastqsanger.gz
	ERR13189371_1.fastq.gz	Fastqsanger.gz

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Primary human CD34+ cells for modeling ETO2-GLIS2 leukemogenesis - Bulk RNAseq

Project description:Several fusion oncogenes showing a higher incidence in pediatric acute myeloid leukemia are associated with heterogeneous megakaryoblastic and other myeloid features. Here we addressed how developmental mechanisms influence human leukemogenesis by ETO2::GLIS2, a hallmark of dismal prognosis. We induced expression of ETO2::GLIS2 in primary human fetal and cord blood CD34+ hematopoietic cells and obtained bulk transcriptomes after in vitro cultures or in vivo engraftment and leukemia development in immunodeficient recipients (NSG or NSG-SGM3=NSG-S).

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Primary human CD34+ cells for modeling ETO2-GLIS2 leukemogenesis - ATACseq

Project description:Primary human CD34+ cells for modeling ETO2-GLIS2 leukemogenesis - ATACseq

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Primary human CD34+ cells for modeling ETO2-GLIS2 leukemogenesis - Bulk RNAseq

Project description:Primary human CD34+ cells for modeling ETO2-GLIS2 leukemogenesis - Bulk RNAseq

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Primary human CD34+ cells for modeling ETO2-GLIS2 leukemogenesis - Single cell RNAseq

Project description:To characterize the consequences of cytokine stimulation, we compared ET02-GLIS2 expressing cells from different origins by single cell transcriptomes (scRNAseq). We analysed FL and CB ET02-GLIS2-expressing CD34+ progeny after 7 days in vitro, human cells from diseased NSG (FL- and FBM-derived) and NSGS (CB- and FL-derived) recipients.

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Identification of ETO2-GLIS2 transcriptional targets

Project description:Acute megakaryoblastic leukemia (AMKL) is a subtype of leukemia primarily diagnosed in childhood and generally associated with poor prognosis. Genetic alterations found in de novo childhood AMKL include the OTT-MAL fusion, MLL and NUP98 fusions and the recently identified ETO2-GLIS2 fusion that involves two transcriptional regulators. In order to identify ETO2-GLIS2 target genes, we performed two approaches: 1-Ectopic expression of ETO2-GLIS2, ETO2, GLIS2 and OTT-MAL in HEL cells, which does not endogenously express AMKL fusion oncogenes. Transduced cells marked by expression of the GFP were sorted by flow cytometry 24 hours after transduction and RNA was extracted with the Qiagen Rneasy kit including DNase treatment. 2-Expression of a small peptide (NC128), which interferes with the dimerization and cofactor recruitment by the ETO2-GLIS2 fusion, within MO7E cells derived from an AMKL patient and expressing endogenously the ETO2-GLIS2 fusion. Transduced cells marked by expression of the GFP were sorted by flow cytometry 7 days after transduction and RNA was extracted with the Qiagen Rneasy kit including DNase treatment. After quantification of biological replicates, and quality control (Bioanalyser, Agilent), RNA were hybridized on Agilent arrays as indicated below.

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Identification of ETO2-GLIS2 targets by ChIP-sequencing

Project description:Acute megakaryoblastic leukemia (AMKL) is a subtype of leukemia primarily diagnosed in childhood and generally associated with poor prognosis. Genetic alterations found in de novo childhood AMKL include the OTT-MAL fusion, MLL and NUP98 fusions and the recently identified ETO2-GLIS2 fusion that involves two transcriptional regulators. In order to identify ETO2-GLIS2-bound regions as well as the chromatin landscape in human AMKL cells, we performed ChIP-seq analyses in AMKL MO7E cell line and in AMKL patient derived cells. Since existing GLIS2 antibodies could not successfully pull-down ETO2-GLIS2, we introduced the GFP at the endogenous GLIS2 loci in the MO7E cell line using a CRISPR/Cas9 approach to obtain physiological expression of a GFP-tagged ETO2-GLIS2 (MO7e-KI cell line). ChIP-seq were performed using antibody against GFP and ETO2 to identify ETO2-GLIS2-bound regions, against ERG to investigate colocalization and against chromatin marks to highlight repressed (H3K27me3) and active (H3K4me3: promoters; H3K27Ac, H3K4me1: enhancers) regions.

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Project description:To characterize the molecular consequences of ETO2-GLIS2 expression in iPSC-derived cells, we performed a transcriptome analysis on control and two ETO2-GLIS2 clones at day18 of differentiation. From the control, the CD41+CD42- and CD41+CD42+ populations were sorted to obtain normal immature progenitors and maturing megakaryocytes. From ETO2-GLIS2-expressing cells, CD41+CD42+ and the aberrant CD41lowCD42low were analyzed.Whole transcriptome sequencing (WTS) was performed using Illumina Nextseq500 platform. cDNA libraries were synthesized from 250 ng total RNA using the TruSeq Stranded mRNA kit (Illumina) following manufacturers’ instructions. Briefly, poly-A containing mRNA molecules were reverse transcribed to double stranded cDNA fragments that were then adenylated at 3' ends and ligated to single-index adapters. PCR-enriched libraries were then quantified by Quant-It picogreen assay (Thermo-Fisher) and sized with the High Sensitivity kit on the 2100 Bioanalyzer (Agilent Technologies). Sequencing was performed at 2x80bp using Illumina Sequencing by synthesis (SBS) technology.

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Profiling of H3K27ac in ETO2-GLIS2 positive AMKL cells and ETO2-GLIS2 binding

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