Project description:Total RNA was analyzed from either uninduced or β-estradiol treated G1E-ER-GATA cells to determine changes in gene expression upon induction of erythroid maturation (treated).
Project description:Alas2 gene encodes the rate-limiting enzyme in heme biosynthesis. CRISPR/Cas9-mediated ablation of two Alas2 intronic cis-elements strongly reduced GATA-1-induced Alas2 transcription, heme biosynthesis, and GATA-1 regulation of other vital constituents of the erythroid cell transcriptome. Bypassing Alas2 function in Alas2 cis-element-mutant (double mutant) cells by providing its catalytic product 5-aminolevulinic acid (5-ALA) rescued heme biosynthesis and the GATA-1-dependent genetic network. We discovered a GATA factor- and heme-dependent circuit that establishes the erythroid cell transcriptome. G1E-ER-GATA-1 WT and double mutant cells were examined. Untreated WT, beta-estradiol-treated WT, beta-estradiol-treated double-mutant, and beta-estradiol/5-ALA-treated double-mutant cells were subjected to RNA-seq.
Project description:The ensemble of Foxo3-regulated genes in the erythroid G1E-ER-GATA-1 cell line was determined by knocking down Foxo3 using siRNA, and measuring genome wide transcription by microarray analysis G1E-ER-GATA-1 cells were treated with control or Foxo3-specific siRNA by nucleofection at t = 0 h and t = 24 h. At t = 24 h, cells were treated with M-CM-^_-estradiol to activate ER-GATA-1. RNA was harvested at t = 48 h and processed for microarray analysis.
Project description:Identification of genes regulated by GATA-1 independent of the cofactor FOG-1. Experiment Overall Design: A conditionally activated FOG-1-binding defective mutant of GATA-1, ER-GATA-1(V205G), was expressed in GATA-1-null G1E cells. Transcipt levels were compared in cells untreated or treated with estradiol to activate the GATA-1 mutant.
Project description:We used microarrays to examine what genes could be regurated by LMO2 in erythroid cells. Comparing expression profile in murine G1E-ER-GATA-1 cells treated with control and Lmo2 siRNA on Agilent array. After siRNA transfection, the cells were treated with b-estradiol for 24h to induce GATA-1-mediated erythroid maturation.
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.
Project description:G1E cells are a Gata-1 erythroid-committed cell line derived from targeted disruption of Gata-1 in embryonic stem cells. The ER4 subclone contains an inducible form of Gata-1 (Gata-1-ER, Gata-1 fused to the estradiol receptor ligand binding domain). We performed transcriptome analysis using this cell line. Estradiol was added to culture medium triggering synchronous and homogenous differentiation. At various time points, RNA was sampled and analyzed using the Affymetrix MG-U74Av2 platform. Three biological replicas (A,B, and C) were performed. The thirty hour time course corresponds to development from the late BFU-E stage through the orthochromatic erythroblast stage.
Project description:We employed a gene complementation strategy combined with microarray screening to identify miRNAs involved in the formation of erythroid (red blood) cells. To search for GATA-1-regulated erythroid miRNAs, we used the Gata-1– erythroblast line G1E. These cells proliferate in culture as immature erythroid precursors and undergo terminal maturation when GATA-1 activity is restored. G1E-ER4 is a sub-line stably expressing an estrogen-activated form of GATA-1 (GATA-1 fused to the ligand binding domain of the estrogen receptor). Treatment of G1E-ER4 cells with estradiol induces a GATA-1-regulated program of gene expression with concomitant cellular maturation. We used a microarray to evaluate the expression of 292 different miRNAs in G1E-ER4 cells at 0 versus 24 hours after GATA-1 activation. Affymetrix gene expression profiling has previously been deposited (GEO accession no. GSE628). Keywords: microRNA analysis of a cell-line model of erythroid maturation Two condition experiment, 3 replicates each (independently grown and harvested) of untreated and estradiol-treated (24hrs) G1E-ER4 cells, which express an estrogen-responsive form of the GATA-1 transcription factor. Each sample is compared to a common reference sample, comprised of an equal mixture of all 6 experimental samples.
