Transcriptional effect of CBFA2T3::GLIS2 oncogenic fusion engineering by CRISPR-Cas9 in iPSC-derived hematopoietic cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 4000
ORGANISM(S): Homo sapiens
SUBMITTER: Thomas MERCHER
PROVIDER: E-MTAB-14188 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2025-07-09 | E-MTAB-14189 | biostudies-arrayexpress
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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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.
2017-05-05 | E-MTAB-4332 | biostudies-arrayexpress
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:These data were generated to investigate the impact on the gene expression of the chimeric transcription factor CBFA2T3-GLIS2 (a.k.a ETO2-GLIS2 and abbreviated here as EG) which is associated with pediatric leukemia (LAM7), to assess the functional roles of several domains of this protein (the ETO2 and GLIS2 moiety) on the gene dysregulation induced by EG through the generation of several mutants (deleted for the NHR2 domain of ETO2: dNHR2 or with a C265G mutation in the GLIS2 moiety: C265G). The different EG forms were cloned into a doxycycline-inducible lentiviral vector and transcriptomes were performed 24 hours post doxycycline induction.
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Project description:The ETO-family transcriptional corepressors, including ETO, ETO2 and MTGR1, are all involved in leukemia-causing chromosomal translocations. In every case, an ETO-family corepressor acquires a DNA-binding domain (DBD) to form a typical transcription factor – the DBD binds to target genes, while the ETO moiety contributes essentially to its transcriptional property. A direct comparative study of these “homologous” fusion transcription factors may clarify their similarities and differences in regulating transcription and leukemogenesis. Here, we performed a side-by-side comparison between AML1-ETO and ETO2-GLIS2, the most common fusion proteins in the M2 and M7 subtypes of acute myeloid leukemia, respectively, by inducible expression of them in U937 leukemia cells. We found that, although AML1-ETO and ETO2-GLIS2 can use their own DBDs (i.e., the Runt domain of AML1 and the zinc finger domain of GLIS2) to bind DNA, they actually share a large proportion of genome-wide binding regions dependent on other cooperative transcription factors such as ETS- and CEBP-family proteins. Functionally, AML1-ETO acts as either transcriptional repressor or activator, whereas ETO2-GLIS2 mainly acts as an activator. The repressor-versus-activator functions of AML1-ETO is determined by the abundance of cooperative transcription factors/cofactors on the target genes. Through these mechanisms, AML1-ETO and ETO2-GLIS2 differentially regulate several key transcription factors that are essential for myeloid differentiation. Indeed, AML1-ETO inhibits myeloid differentiation of U937 cells, whereas ETO2-GLIS2 facilitates it. Taken together, this study is the first direct comparative study between AML1-ETO and ETO2-GLIS2 in the same cellular context, and the results provide new insights into the context-dependent transcriptional regulatory mechanisms that may underlie how these seemingly “homologous” fusion transcription factors exert distinct functions to drive different subtypes of leukemia.
2022-07-05 | GSE207216 | GEO
Project description:The ETO-family transcriptional corepressors, including ETO, ETO2 and MTGR1, are all involved in leukemia-causing chromosomal translocations. In every case, an ETO-family corepressor acquires a DNA-binding domain (DBD) to form a typical transcription factor – the DBD binds to target genes, while the ETO moiety contributes essentially to its transcriptional property. A direct comparative study of these “homologous” fusion transcription factors may clarify their similarities and differences in regulating transcription and leukemogenesis. Here, we performed a side-by-side comparison between AML1-ETO and ETO2-GLIS2, the most common fusion proteins in the M2 and M7 subtypes of acute myeloid leukemia, respectively, by inducible expression of them in U937 leukemia cells. We found that, although AML1-ETO and ETO2-GLIS2 can use their own DBDs (i.e., the Runt domain of AML1 and the zinc finger domain of GLIS2) to bind DNA, they actually share a large proportion of genome-wide binding regions dependent on other cooperative transcription factors such as ETS- and CEBP-family proteins. Functionally, AML1-ETO acts as either transcriptional repressor or activator, whereas ETO2-GLIS2 mainly acts as an activator. The repressor-versus-activator functions of AML1-ETO is determined by the abundance of cooperative transcription factors/cofactors on the target genes. Through these mechanisms, AML1-ETO and ETO2-GLIS2 differentially regulate several key transcription factors that are essential for myeloid differentiation. Indeed, AML1-ETO inhibits myeloid differentiation of U937 cells, whereas ETO2-GLIS2 facilitates it. Taken together, this study is the first direct comparative study between AML1-ETO and ETO2-GLIS2 in the same cellular context, and the results provide new insights into the context-dependent transcriptional regulatory mechanisms that may underlie how these seemingly “homologous” fusion transcription factors exert distinct functions to drive different subtypes of leukemia.
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Project description:Abstract: SR12813 is an experimental cholesterol-lowering drug that reduces intracellular cholesterol through accelerated proteasomal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and is also recognized as a prototypical activator of the pregnane X receptor (PXR, NR1I2). Rifampicin, a clinically used antibiotic, likewise functions as a human PXR agonist. Although PXR-mediated induction of drug metabolism genes has been extensively characterized in hepatocytes and humanized mouse liver, comparatively little is known about the transcriptional effects of these ligands in intestinal and colon cancer cells. Here, we employed RNA sequencing in LS180 colon adenocarcinoma cells to compare transcriptional responses elicited by SR12813 and Rifampicin. LS-180 cells were treated for 48 hours with DMSO, Rifampicin or SR12813.
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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.
2020-04-27 | E-MTAB-7967 | biostudies-arrayexpress
Project description:Profiling of H3K27ac in ETO2-GLIS2 positive AMKL cells and ETO2-GLIS2 binding
| PRJNA543772 | ENA