The Study Of The Effect Of PPP2Cβ On Erythroid Differentiation And The Role Of EDAG In Leukemia Research

Erythropoiesis is tightly regulated at multilevels, among which transcription factors play an important role during the differentiation and maturation of erythrocyte.GATA1 is the best studied erythroid transcription factor. On one hand, the activity of GATA1 can be regulated by posttranslational modification such as acetylation and phosphorylation; On the other hand, GATA1 forms dynamic complex with other transcriptional factors which selectively regulates downstream target genes, then play critical role in erythroid differentiation. In the present study, we focus on two interacting proteins of GATA1, PPP2Cβ and EDAG. We identify PPP2Cβ as a novel regulator of GATA1 transcription activity and a potential positive regulator of erythroid differentiaion. Furthermore, we investigate the role of EDAG in CML(chronic myeloid leukemia) leukemia induced by BCR-ABL and AML(Acute myeloid leukaemia) induced by MLL-AF9. This study composes two parts as follows:First part: PPP2Cβ interacts with GATA1 and postively reugaltes erythroid differentiation.Previously, we have constructed an interaction network involved hematopoietic transcriptional factors using yeast two hybridization. In this network, the catalytic subunit of protein phosphatase 2A(PPP2Cβ) is a novel interacting protein with GATA1, indicating PPP2Cβ might be involved in hematopoietic differentiation. First,we detected the expression profile of PPP2Cβ during erythroid differentiation ofhuman cord blood CD34+ cells induced by erythropoietin(EPO). Accompanied by erythroid differentiaiton, PPP2Cβ is upregulated both at mRNA and protein levels and at the later stage of differentiation, the expression level of PPP2Cβ returns to basic level. These data demonstrate that PPP2Cβ might play key role in early stage of erythroid differentiation.Furthermore, we constructed Flag tagged PPP2Cβ expression vector, and confirmed the interaction between GATA1 and PPP2Cβ in cells by co-immunoprecipitation. Also, we have confirmed that PPP2Cβ binds to the N-terminal zinc finger of GATA1. Furhtermore, using dual-luciferase reporter gene assay we found that PPP2Cβenhances the transcriptional activity of GATA1 in a dose-dependent manner. The transcription of downstream genes of GATA1, inculding EKLF, P45, Gfi-1b and GAP which is verified were also induced by PPP2Cβoverexpression. In addition, overexpression of PPP2Cβ in K562 cells led to spontaneous differentiate toward erythroid.These results demonstrate that PPP2Cβ can regulate the activity of GATA1, and serve as a novel regulator of erythropoiesis.Second part: The role of EDAG in BCR-ABL-induced CML and MLL-AF9-induced AML.Erythroid Differentiation Associated Gene(EDAG) is a firstly isolated and cloned by our laboratory, which is specially expressed in hematopoietic tissues and involved in regulating the hematopoietic cells proliferation, differentiation of and lineage commitment balance of HSC. Our recent study reveal that EDAG can interact with GATA1, recruit p300 and enhance the acetylation level and activity of transcriptional regulation of GATA1. The dynamic complex of these three molecular can selectively activate genes associated with eryrhroid differentiation and thus promote erythropoietic differentiation. Furthermore, we discover that EDAG may berelated to pathogenesis of leukemia. In this part, we investigated the role of EDAG in BCR-ABL induced CML and MLL-AF9 induced AML.First, we packaged retrovirus particles of BCR-ABL, and get high efficiency of infection. Also, we established BCR-ABL induced CML mouse model. The expression of EDAG is upregulated in the umbilical cord blood derived CD34+infected with BCR-ABL retrovirus. Then we infected the bone marrow cells of EDAG knockout mice or wild type mice to perform bone marrow transplantation. The CML onset time of recipient mice transplanted with EDAG knockout bone marrow cells significantly later than that transplanted wild type mice bone marrow. The white blood counts were much lower than control. These data indicate EDAG plays an important role in BCR-ABL induced CML.Moreover, we examined the effect of EDAG on AML cell proliferation and invasion in vivo using MLL-AF9 transgenic mice. EDAG is down-regulated in bone marrow and spleen of AML mice. Then we transplanted spleen cells of MLL-AF9 transgenic mice into EDAG knockout mice or wild type mice, and the white blood counts were measured. And the data suggest that the counts of peripheral blood between two groups showed no obvious difference.In conclusion, we identify PPP2Cβ as a novel regulator of GATA1 activity and erythropoietic differentiation. And the hematopoietic transcriptional regulator EDAG plays an important role in the development of CML induced by BCR-ABL.