Functional Analysis of FOXO3A Involved in Erythroid Differentiation

Abstract 4731

FOX (Forkhead box) proteins are a family of transcription factors that emerged as playing an important role in the embryonic development, cell cycle, carbohydrate and fatty acid metabolism and immune response. It was found that FOXO3A (also known as FOXO3) involved in erythroid differentiation, yet the mechanism for regulating hematopoietic stem cells (HSCs) differentiation is unknown. We analyzed the dynamics of genome-wide transcriptome (mRNA-Seq) of human undifferentiated embryonic stem cells (HESC), erythroid cells derived from ES cells (ESER), human fetal erythroid liver cells (FLER) and peripheral CD34⁺derived erythroid cells (PBER) using high throughput sequencing technology. The transcriptome analysis showed that FOXO3 was barely expression in HESC while was observably up-regulated in ESER. However, FOXO3 was down-regulated in FLER and PBER compare with ESER, the erythroid cells at early developmental stage. We presumed that FOXO3 plays an important role in primitive erythropoiesis and built up the interactions network in which FOXO3 acts as a central node by Gene Ontology (GO), correlation analysis and Ingenuity Pathways Analysis (IPA). In addition, we analyzed the profiles of histone methylation in the four types of cells by ChIP-Seq to study the chromatin conformation in the vicinity of FOXO3. More histone 3 lysine 4 (H3K4) trimethylation was found near the promoter region of FOXO3 in ESER compared with the other cells, which is coincided with the mRNA-seq results.

We performed a series of experiment to identify the roles of FOXO3 in regulating erythroid differentiation. The results showed that the expression level of ε and γ globin were up-regulated in FOXO3-over-expressed 293T and Hela cells and the expression level of FOXO1 and CAT in predicted network were increased by quantitative real-time PCR detection. In addition, when FOXO3 knocked down in K562 cells, the expression level of ε and γ globin were down-regulated. The expression level of CAT, BCL2L1 and other factors in predicted network, were also decreased. These results indicate FOXO3 plays an important role in globin expression and identify the credibility of our predicted networks in which FOXO3 acts as a central node. FOXO3 binding sites (GTAAACA or ATAAACA) were predicted on the upstream of CAT and BCL2L1. We are trying to prove CAT or BCL2L1 is a direct FOXO3 target in vitro and in vivo.

In conclusion, we have demonstrated FOXO3 plays a key role in erythroid differentiation and globin expression. We will further determine the enriched profiles of FOXO3 by ChIP-seq in HESC, ESER, FLER and PBER to find more targets of FOXO3. Since the zebrafish is a powerful model system for investigating vertebrate hematopoiesis. We will identify the role of Foxo3b, the homologous gene of human FOXO3, in erythroid differentiation and study the dynamic transcriptomes of Foxo3b morphants in zebrafish. We are trying to make a whole picture to elaborate the molecular mechanism of FOXO3 involved in regulation of erythroid differentiation.