A Systems Approach Identifies Essential FOXO3 Functions at Key Steps of Terminal Erythropoiesis
Fig 4
Impaired enucleation-related gene transcription in Foxo3-/- bone marrow erythroblasts.
(A) WT and Foxo3-/- bone marrow TER119+ erythroblasts were analyzed for DRAQ5 staining by flow cytometry. The percentage of enucleated TER119+ cells is shown (lower panel). Results are mean ± SEM of n = 3; one representative of four different experiments is shown. (B) QRT-PCR expression analysis by Fluidigm microfluidics technology of enucleation-related genes in WT and Foxo3-/- Gates I to IV bone marrow erythroblasts. Quantification of target genes is normalized to β actin. Results are mean ± SEM of 3 cDNAs. (C) FOXO3 occupation of Mxi1 (left) and Riok3 (right) regulatory regions as determined by ChIP. Enrichment of putative FOXO3 DNA binding regions in Mxi1 and Riok3 promoters was analyzed by qPCR and compared to regions with no known FOXO3 binding sites. Values were normalized to Ct values from total input. One representative of two different experiments is shown. NS Not specific; DBE DNA binding element. *P < 0.05 **P < 0.01 ***P < 0.001, Student’s t test.