Study Of The Molecular Mechanism Of EDAG In Hematopoietic Cell Differentiation

Erythroid differentiation-associated gene (EDAG), a hematopoietic tissue-specific transcription regulator, plays a key role in maintaining the homeostasis of hematopoietic lineage commitment. However, the mechanism and genes regulated by EDAG remain unknown. In this study, we investigate the function and molecular mechanism of EDAG through three aspects.First, we studied the molecular mechanism of EDAG in hematopoietic cell differentiation by identifying genes regulated by overexpressing EDAG in a myeloid cell line 32D. Overexpression of EDAG in 32D cells led to an erythroid/ megakaryocytic phenotype. Using a genome-wide microarray analysis and a two-fold change cutoff, we identified 332 genes with reduced expression and 288 genes with increased expression. Among up-regulation genes, transcription factor GATA-1 and its target genes including EKLF, hemoglobin, NF-E2, Gfi-lb etc. were increased. While monocyte differentiation antigen CD 14 and mast cell surface glycoprotein GP49A were decreased. Silencing of EDAG by RNA interference in K562 cells resulted in down-regulation of GATA-1 and its target genes. These results suggested that EDAG functions as a positive regulator of erythroid/megakaryocytic differentiation in 32D cells associated with the induction of GATA-1 and its target genes.Second, we focused on indentifying the function and structure of EDAG. Using the dual-luciferase reporter assay, Gal4DBD-EDAG fusion protein was found as a transcriptional repressor in different mammalian cells. However, two transactivation domains (124-184aa and 384-484aa) was characterized and showed significant transcriptional activation activity. Co-expression of transcriptional repressor CtBP1 repressed the transactivaiton activity of two transactivation domains. Furthermore, two serine sites (S123 and S381) can be phosphorylated under treatment with PMA, but mutation of these amino acids had no effect on the activity of EDAG. These results indicated that EDAG both has transactivation and repression activity, and the mechanism by which it regulates gene expression is more complicated. Third, we characterized NPM as a physiological binding partner of EDAG. EDAG was shown to interact with the N-terminal (1-187aa) of NPM through its N-terminal (1-124aa) region, and stabilized NPM protein. During PMA-induced K562 megakaryocytic differentiation, overexpression of EDAG prevented the down-regulation of NPM proteins, while knockdown of EDAG enhanced the degradation of NPM.