Functional Analysis Of FOXO3in Erythroid Differentiation

Erythroid differentiation and development is an extremely complex and precise biological process. Hemoglobin is the protein specifically expressed in erythroid cells that carries oxygen. The expression of its member varies at different development stages. An abnormal form of hemoglobin result in heritable blood disorders such as thalassemia. It is of theoretical and clinical importance for the regulatory mechanisms of erythropoiesis and the molecular cures of thalassemia to study the regulation of hemoglobin expression. We assumed that FOXO3plays an important role in primitive erythropoiesis by integratively analyzing the dynamics of genome-wide transcriptome (mRNA-Seq and miRNA-Seq), DNaseI hypersensitivity (DNase-Seq) and histone methylation of H3K4me3(ChIP-Seq) in undifferentiated human embryonic cells (HESC) as well as erythroid cells at embryonic(ESER), fetal (FLER) and adult (PBER) stage using high throughput sequencing technology.Globin expression was significantly increased while erythroid-specific transcription factor GATA1and KLF1expression was not after over-expressed FOXO3in non-erythroid cells:293T and Hela cells. When inhibited FOXO3expression in two erythroid cells chronic myelogenous leukemia cell line (K562) and erythroleukemia cell line (TF-1), we found that globin expression was striking decreased while the expression of GATA1and KLFl expression was different between K562and TF-1cells with no changes in the former and reduced in the latter. These results suggest that FOXO3can regulate globin expression and promote erythroid differentiation. BTG1was predicted as a target of FOXO3as there is a FOXO3binding site in the upstream of BTG1by using Ingenuity Pathways Analysis (IPA) and TFBS sequencing searching. We further validated the interaction between BTG1and FOXO3using Dual-Luciferase Reporter Assay System. In addition, we screened the miRNAs that regulate FOXO3from miRNA-Seq data and validated the suppression function of miR-200on FOXO3. These results indicate that the regulation direction of miR-200b, FOXO3, and BTG1may play particular role in erythroid differentiation.Zebrafish is a good vertebrate model organism to study the hematopoiesis by reason of the highly conservative hematopoietic system to human. Compared with zebrafish that was injected with only morpholinos, the expression of globin and Gatal genes as well as the expression of haemoglobin was repressed and the erythroiod development was abnormal in zebrafish that was injected with Foxo3b morpholinos. These phenotype variations were similar to those in both human cells K562and TF-1with FOXO3knockdown. We infer that FOXO3/Foxo3b plays an important role in erythroid differentiation in vertebrate.