EDAG Enhances GATA-1Protein Stability And Plays Key Role In Erythroid Differentiation

EDAG is a hematopoietic tissue-specific transcription regulator that plays a keyrole in maintaining the homeostasis of hematopoietic lineage commitment, is involvedin the regulation of hematopoietic cell proliferation, differentiation and apoptosis.Previous study suggest that EDAG interacts with GATA-1and NPM1, but the roles ofthese interactions remain unknown. In the present study, we investigated theseinteractions in detail.In the present study, we confirmed the interation between EDAG and GATA-1,mapping the binding domain between them. Then we found that over-expression ofEDAG was able to enhance the protein stability of GATA-1, and then expanding thehalf-life of GATA-1turnover rate in HEK293cells. In K562cells, under CHXtreatment, the stability of GATA-1protein was enhanced by EDAG over-expression,while knock down of EDAG by lentivirus-mediated siRNA result in an increaseddegradation rate of GATA-1in K562cells. During PMA-induced K562megakaryocytic differentiation, overexpression of EDAG prevented thedown-regulation of GATA-1proteins, while knockdown of EDAG accelerated thedown-regulation of GATA-1. EDAG also increased DNA-binding activity andtranscriptional activity of GATA-1. During Epo-induced erythroid differentiation ofCD34+cells, knockdown of EDAG inhibited the nucleus localization of HSP70,induced the activation of Caspase3, and then inhibited the up-regulation ofendogenous GATA-1.The erythroid differentiation of CD34+cells induced by EPOwas inhibited by EDAG knockdown, suggesting a critical role of EDAG in erythroiddifferentiation.NPM1(Nucleophosmin or B23), a ubiquitous nucleolar phosphoprotein, has beenproved to be a candidate binding protein of EDAG and primary study suggests thatEDAG enhances the protein stability of NPM1. In the present study, usingco-immunoprecipitation, we characterized EDAG as a physiological binding partnerof NPM1, and EDAG interacts with the N-terminal (118-187aa) of NPM1through itsN-terminal (1-124aa) region. Under CHX treatment, the stability of NPM1proteinwas enhanced by EDAG overexpression, while knockdown of EDAG bylentivirus-mediated siRNA result in an increased degradation rate of NPM1in K562 cells. During PMA-induced K562megakaryocytic differentiation, overexpression ofEDAG prevented the down-regulation of NPM1proteins, while knock down ofEDAG accelerated the down-regulation of NPM1. EDAG deletion mutant lacking thebinding domain with NPM1lost the ability to stabilize NPM1protein. Furthermore,knockdown of EDAG in K562cells led to increased cell apoptosis induced byimatinib, and re-expression of NPM1attenuated the increased apoptosis. These resultssuggest that EDAG enhances the protein stability of NPM1through binding to NPM1,which plays a critical role in the anti-apoptosis of leukemia cells.We further identified EDAG as a novel substrate of p300and p300acetylatedEDAG protein. Three acetylated sites (K189, K327, K341) were identified by co-IPwith MS/MS. Interestingly, EDAG acetylation can increase its protein stability, andenhance the interaction between EDAG and GATA-1.In summary, we find that EDAG can regulate erythroid differentiation throughincrease the GATA-1stability, and EDAG play a role in the drug resistance ofleukemia chemotherapy by increase NPM1protein stability. EDAG protein stability isregulated by acetylation of p300.