Study Of The Effect Of EDAG On The Differentiation And Proliferation Of Human CB CD34~+Cells

EDAG is a transcriptional regulator which was identified from fetal liver usingRDA (Representational Difference Analysis, RDA) and specifically expresses inhematopoietic tissue. The expression level of EDAG is high in HSPC (HematopoieticStem/Progenitor Cells, HSPCs) and erythroid progenitors, while it is low in matureblood cells. Previous studies suggest that EDAG promotes the proliferation ofhematopoietic cells, inhibits cell apoptosis and regulates cell differentiation, playing akey role in maintaining the homeostasis of hematopoietic lineage commitment. As apositive feedback regulator of GATA1, which is a key transcription factor in erythroiddifferentiation, EDAG overexpression increases the expression level of GATA1andits target genes, and then leads to an erythroid phenotype in32D cell line. However,the effect of EDAG on the proliferation, differentiation and reconstitution ability ofhuman HSCs (hHSCs) remains unclear. In this study, the effects of EDAG on HSCswere investigated in human core blood (CB) CD34~+cells.Firstly, EDAG promotes erythroid differentiation in vitro. EDAG expression inhuman bone marrow HSPC was first examined. HSCs, CMPs, CLPs and MEPs wereisolated from bone marrow, and the results suggested that EDAG expression in CLPswas approximately100-fold less compared to the expression in HSCs, whereasEDAG expression in CMPs and MEPs was5-8-fold greater than in HSCs.Examination of CB CD34~+cells and cultured burst-forming unit-erythroid (BFU-E)and colony-froming unit-erythroid (CFU-E) colonies showed that EDAG expression,similar to GATA1expression, was up-regulated in BFU-E and CFU-E cells. Inaddition, EDAG expression was significantly up-regulated in CD34~+cells subjectedto EPO-induced erythroid differentiation. To investigate the effect of EDAG onerythroid differentiation, EDAG overexpressing and RNAi lentivrus vectors wereconstructed then lentivrus particles were packaged. The infection efficiency of the lentivirus on human CB CD34~+cells was about40%. In erythroid-inducing liquidculture, EDAG overexpression increased the percentage of CD71~+GPA~+cells andpositive benzidine-staining cells. Accordingly, knockdown of EDAG decreased thepercentage of CD71~+GPA~+cells and positive benzidine-staining cells. In semisolidculture, the overexpression of EDAG increase the CFC (Colony-Forming Cell)number and the proportion of BFU-E whereas knockdown of EDAG greatly reducedthe CFC numbers and the proportion of BFU-E. EDAG was a positive feedbackregulator of GATA1, and overexpression of EDAG or GATA1could increase themRNA level of each other, and further increase the level of GATA1-targetd erythroidgenes. In contrast, knockdown of EDAG decreased the level of GATA1and its targeterythroid genes. These results suggested that EDAG promotes the erythroiddifferentiation and maturation of CD34~+cells. The mechanism of EDAG promotingerythroid differentiation was further investigated. Two EDAG deletion mutants (1-124aa and124-484aa) were transduced into CD34~+cells, and erythroid differentiationwas investigated. The EDAG mutant that lacks the GATA1binding domain (124aa-484aa) was unable to accelerate erythroid differentiation. Interestingly, thedeletion mutant containing the GATA1-binding domain (1-124aa) but lacked thep300binding domain showed a clear inhibitory effect on erythroid differentiation.These result suggest that EDAG regulates erythroid differentiation dependent on theformation of EDAG/GATA1/p300complex. Furthermore, a specific inhibitor of theacetylation transferase of p300treatment led to a dramatic inhibition of erythroiddifferentiation, and in the presence of C646, forced expression of EDAG failed toenhance erythroid differentiation, indicating that the regulation of erythroiddifferentiation by EDAG is mediated by p300. Moreover, overexpressing EDAGpromoted cell proliferation and inhibited apoptosis after EPO deprivation duringerythroid differentiation, whearas knockdown of EDAG delayed the proliferation andincreased apoptosis. To determine whether EDAG regulates the transcription ofGATA1target genes, cDNA microarray analysis was performed after EPO inductionin CD34~+cells infected with control virus or EDAG shRNA lentivirus. A total of1527genes were down regulated, and1622genes were upregulated upon EDAGknockdown compared with the control. EDAG knockdown led to the down-regulationof the majority of erythroid genes that are activated by GATA1, such as gata1, hbb,eklf, bcl11a, gypa, and slc4a1. However, the transcription levels of most GATA1-repressed genes, such as myc, myb, kit, and gata2, did not significantlychange in EDAG knockdown cells. Surprisingly, a subset of the genes inhibited byGATA1, such as itm2a, dlc2, bzw2, spint2and kpnb1, were also found to bedownregulated in EDAG knockdown cells, suggesting that EDAG may regulate theexpression of these genes independently of GATA1. In addition to the downregulationof erythroid genes, EDAG knockdown resulted in the upregulation of myeloid genesand lymphoid genes, suggesting that EDAG has a negative regulatory effect on theexpression of these genes.Secondly, EDAG promotes erythropoiesis in vivo. The model ofimmunodeficient mouse transplantation was constructed after comparing the differentmouse strains, irradiation conditions and CD34~+infection strategies, and then it wassuggested that NOD/SCID/IL2nullmouse receiving2times X-Ray irradiation with atotal dose of2.5Gy and then an injection of lentivrus-infected CD34~+cells, whichwere freshly isolated and subjected to2times of lentivirus infection within72hoursafter isolated, showed higher engraftment and better erythropoiesis efficiency. CBCD34~+cells infected EDAG overexpressing or control lentivrus were transplantedinto irradiated NOD/SCID/IL2nullmouse, and the human-derived cells in bonemarrow were analyzed4weeks after transplantation. Consistent with the in vitroresults, the cells that over-expressing EDAG and reconstructed in mice gave rise to asignificantly higher proportion of human erythroid cells (GFP~+CD45-CD71~+GPA~+,median:9.62%, n=5) compared to cells transduced with control virus (median:2.98%,n=5). This finding suggests that EDAG over-expression also promotes the generationof erythroid cells in vivo. Furthermore, EDAG overexpression increased thepercentage of CD11b~+cells and CD34~+cells, which indicated that EDAG alsopromotes myelopoiesis and plays a role in the self-renewal of HSCs.Thirdly, EDAG promotes cell proliferation and inhibits apoptosis of CD34~+cells.In the liquid expansion medium, EDAG overexpression had no effect on lineagecommitment except increased CD11b~+cells and CD34~+cells, suggesting that EDAGpromotes myeloid differentiation and that EDAG participates in the regulation ofself-renewal of HSCs. In addition, EDAG overexpression promoted cell proliferation,inhibits cytokine-deprivation-inducing apoptosis and sustained cells in cell cycle,which were in accordance with the results that EDAG knockdown led to decreasedproliferation, more apoptosis cells after cytokine deprivation and more cells in G0-phase. These results indicated that EDAG regulates the proliferation, survival andcell cycle progress of HSCs.Taken together, EDAG accelerates erythroid differentiation both in vitro and invivo, depending on the formation of EDAG/GATA1/p300complex. In HSCs, EDAGpromotes cell proliferation, enhances the survival and sustains the cells in cell cycle.These finding suggest that EDAG plays a key role in the regulation of self-renewaland differentiation of HSCs.