Mi2M-CM-^_ knockdown in M-CM-^_-estradiol-treated (24 h) G1E-ER-GATA-1 cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Andrew DeVilbiss
PROVIDER: E-GEOD-48188 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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.
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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.
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Project description:Analysis of erythroid differentiation using Gata1 gene-disrupted G1E ER4 clone cells. Estradiol addition activates an ectopically expressed Gata-1-estrogen receptor fusion protein, triggering synchronous differentiation. 30 hour time course corresponds roughly to late burst-forming unit-erythroid stage (t=0 hrs) through orthochromatic erythroblast stage (t=30 hrs). Experiment Overall Design: G1E ER4 cells cultured in G1E medium were treated at 6 time points with estradiol to initiate erythroid differentiation by activating Gata1 transcription factor and total RNAs from treated cells were extracted for microarray experiment. The erythroid differentiation status was confirmed by cell pellet color and expression of microRNA miR451. The design was similar to an earlier studies (Welch, J. J., Watts, J. A., Vakoc, C. R., Yao, Y., Wang, H., Hardison, R. C., Blobel, G. A., Chodosh, L. A., and Weiss, M. J. (2004)). Global regulation of erythroid gene expression by transcription factor GATA-1. Blood 104, 3136-3147), except that a more recent version of Affymetric chip was used to acheive greater transcriptome coverage.
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