Embryonic erythropoiesis and hemoglobin switching require transcriptional repressor ETO2 to modulate chromatin accessibility and looping [RNA-Seq 2]
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE156306 | GEO | 2020/08/25
REPOSITORIES: GEO
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Project description:ETO2 functions as a transcription repressor and is required for the embryonic erythropoiesis and the hemoglobin switch. To gain insight into ETO2 regulatory function during human erythropoiesis, we performed RNA-seq for WT and ETO2 KO K562 cells and found that up-regulated genes upon ETO2 loss in human cells included many markers of mature erythroid cells EPB42, ALAS2, GYPA and SLC25a37. Notably, the α-globin genes (HBA1, HBA2 and HBZ) and embryonic and fetal β-globin genes (HBE1, HBG1, and HBG2) were significantly increased after deletion of ETO2. By contrast, deletion of ETO2 down-regulated the transcription factor genes (ETS1, KLF8 and SOX6) which play a negative role in globin gene expression and hemoglobin synthesis. To further explore different domain function of ETO2, we have analyzed our RNA-seq data from domain deletion cell lines compared to the cell line expressing wild type ETO2. Generally, 702 genes were found to co-regulated by three domain function of ETO2. Interestingly, the regulation of fetal globin genes, erythroid regulator (SOX5) as well as epigenetic factor (HDAC7) is required by three domain function of ETO2. During mouse embryonic erythropoiesis, our FACS-sorted and normal RNA-seq data in E14.5 fetal liver cells indicated that eto2 promoted a critical developmental transition and played an important role in globin switch from embryonic to adult β-globin transcription since its function is essential for key regulators (PU.1, BCL11A and ZBTB7A) and globin genes (Hbb-y and Hba-x) regulation.
2020-08-25 | GSE142226 | GEO
Project description:The Ldb1/GATA-1/TAL1/LMO2 complex mediates long range interaction between the β-globin locus control region (LCR) and gene in adult mouse erythroid cells but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated human NLI (Ldb1 homologue) complex occupancy and chromatin conformation of the β-globin locus in human erythroid cells. In addition to the LCR, we find robust NLI complex occupancy at a site downstream of the Aγ-globin gene, within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing β-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members together with co-repressor ETO2 and γ-globin repressor BCL11A. The LCR and β-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is re-activated by cytokines in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. Thus, alternative NLI complexes mediate γ-globin transcription or silencing through long range LCR interactions involving an intergenic site of non-coding RNA transcription and ETO2 is critical to this process. ChIP-chip of GATA-1, NLI, and pol II in cell lines
2012-01-05 | GSE30047 | GEO
Project description:The Ldb1/GATA-1/TAL1/LMO2 complex mediates long range interaction between the β-globin locus control region (LCR) and gene in adult mouse erythroid cells but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated human NLI (Ldb1 homologue) complex occupancy and chromatin conformation of the β-globin locus in human erythroid cells. In addition to the LCR, we find robust NLI complex occupancy at a site downstream of the Aγ-globin gene, within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing β-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members together with co-repressor ETO2 and γ-globin repressor BCL11A. The LCR and β-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is re-activated by cytokines in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. Thus, alternative NLI complexes mediate γ-globin transcription or silencing through long range LCR interactions involving an intergenic site of non-coding RNA transcription and ETO2 is critical to this process. ChIP-chip of GATA-1, NLI, and pol II in cell lines Comparison of GATA-1, NLI, and pol II binding on chromatin. 2 replicates for each protein; ChIP and input DNA for each replicate
2012-01-05 | E-GEOD-30047 | biostudies-arrayexpress
Project description:We used microarrays to examine what genes could be regulated by ETO2 in erythroid cells. Comparing expression profile in murine G1E-ER-GATA-1 cells treated with control and ETO2(Cbfa2t3) siRNA on Agilent array. After siRNA transfection, the cells were treated with b-estradiol for 24h to induce GATA-1-mediated erythroid maturation.
2010-09-25 | E-GEOD-24333 | biostudies-arrayexpress
Project description:Krueppel-like factor 1 (KLF1) is a major transcription factor, which regulates the γ-globin to β-globin gene switch during erythropoiesis. The goal of this study was to investigate genome-wide transcriptome expression pattern in the presence of KLF1 knockdown to discover targets of KLF1 regulation involved in γ-globin silencing. To achieve KLF1 gene silencing, we stably transfected human primary erythroid cells generated from CD34+ cells, with a short hairpin lentiviral vector carrying a shKLF1 (short hairpin RNA for KLF1 loop) and a puromycin resistance gene. A set of three shKLF1 were designed uusing siDesign Center (Dharmacon) with the designations: 1) shKLF1-1, located in zinc finger at 3'-terminal; 1) shKLF1-2 located in Transcription Factor II H, and 3) shKLF1-3 located in 5’-terminal without a function region. After lentiviral packaging and transduction into CD34+ cells, each expression virus with encoding GFP, puromycin resistance fragment, and a loop consists of the KLF1 knock-down domain. The three shKLF1 vectors (shKLF1-1, shKLF1-2, shKLF1-3), along with scrambled control (shScr) was used to transduce human adult CD34+ stem cells isolated from peripheral blood of healthy adults. The cells were grown in one-phase liquid culture system as previously published by our group (Li B et al. Characterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturation. BMC Genomics. 2012; 13:153). At day-15 in the 28-day culture period, by real-time PCR and Western blot analysis we found KLF1 gene silencing mainly for shKLF-1 and shKLF-2; however, we isolated total RNA from the three stable lines for RNA-seq analysis.
2021-05-12 | GSE174276 | GEO
Project description:microRNA profiling of human K562 cells comparing control or ETO2-overexpressed cells Two-condition experiment, K562-pcDNA vs. K562-pcDNA-ETO2, Biological replicates: 1, 1 pcDNA, 1 pcDNA-ETO2, independently.
2014-10-01 | E-GEOD-51485 | biostudies-arrayexpress
Project description:microRNA profiling of human K562 cells comparing control or ETO2-overexpressed cells
2014-10-01 | GSE51485 | GEO
Project description:The Krüppel-like factors, KLF1 and KLF2, positively regulate embryonic β-globin expression, and have additional overlapping roles in embryonic (primitive) erythropoiesis. KLF1-/-KLF2-/- double knockout mice are anemic at embryonic day 10.5 (E10.5) and die by E11.5, in contrast to single knockouts. To investigate the combined roles of KLF1 and KLF2 in primitive erythropoiesis, expression profiling of E9.5 erythroid cells was performed. A limited number of genes had a significantly decreasing trend of expression in wild-type, KLF1-/- and KLF1-/-KLF2-/-. Among these, c-myc emerged as a central node in the most significant gene network. c-myc expression is synergistically regulated by KLF1 and KLF2, and both factors bind the c-myc promoters. To characterize the role of c-myc in primitive erythropoiesis, ablation was performed specifically in mouse embryonic proerythroblast cells. After E9.5, these embryos exhibit an arrest in the normal expansion of circulating red cells and develop anemia analogous to KLF1-/-KLF2-/-. In the absence of c-myc, circulating erythroid cells do not show the normal increase in α- and β-like globin expression, but interestingly, have accelerated erythroid maturation, between E9.5 and E11.5. This study reveals a novel regulatory network by which KLF1 and KLF2 regulate c-myc, to control the primitive erythropoietic program. Timed-pregnant KLF1+/-, KLF1+/- KLF2+/- females were anesthetized and sacrificed. E9.5 yolk sacs were dissected from the embryo, cryoprotected in 20% sucrose in PBS and frozen in OCT media. A small portion of the embryo tail was used for PCR genotyping. Eight micron frozen yolk sac sections were obtained and laser capture microdissection (LCM) was used to isolate primitive erythroid precursors. For each biological replicate, 2 to 4 yolk sacs from 2 different litters were used. Total RNA was isolated from 8 different wild-type, 3 KLF1-/-, 3 KLF1-/- KLF2-/- erythroid samples and hybridized to Affymetrix 430 A 2.0 microarrays.
2012-03-12 | E-GEOD-36427 | biostudies-arrayexpress
Project description:ETO2 (CBFA2T3) functions as a transcriptional corepressor, which can directly bind to E-protein family of transcriptional factors. Translocation of ETO2 has been implicated in multiple leukemogenic pathways. ETO2 also plays an important role in self-renewal, proliferation, and expansion of hematopoietic stem/progenitor cells (HSPCs). Here we report the genomic binding sites of ETO2 in human cord-blood derived CD34+ HSPCs, Kasumi-1 t(8;21) AML cells, and U937 AML cells. All ChIP-Seq assays were performed with an ETO2 specific antibody that does not recognize other ETO/MTG family proteins (ETO or MTGR1).
2019-05-29 | GSE126953 | GEO
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