Erythropoietin (Epo) activates multiple intracellular signal transduction pathways, including StatS, PI -3kinase/Akt, and Shc/Ras/ MARK, that interact with key erythroid transcription factors including GATA1 andFOG to prevent apoptosis of CFU-E progenitors, trigger 3-5 terminal cell divisions, and induce multiplegenes essential for erythrocyte formation. Most erythroid- specific genes induced by StatS, GATA1, and FOGare unknown. Here we will use a combination of chromatin immunoprecipitation with mouse promotermicroarrays (ChlP-on-chip) to identify promoters bound by Stat5b, GATA1, and FOG in erythroid progenitorsand subsequent stages of erythroid differentiation purified from mouse fetal livers. In parallel we willdetermine the mRNA expression profiles of erythroid progenitors at different stages of terminal erythroiddifferentiation and determine genes directly and indirectly regulated by the Stat5 and Akt signaling pathways.To this end we will obtain transcriptional profiles of erythroid cells isolated from Stat5a-/-b-/- embryos;ectopic reexpression of Stat5b can confirm certain genes as direct targets of Stat5. In parallel we willexamine the transcriptional profiles in primary erythroid cells deficient in EpoR- mediated Akt activation oroverexpressing a constitutively active Akt kinase. Recently we showed that erythroid progenitors mustadhere to fibronectin via alpha4beta1, or alpha5beta1 integrin in order to undergo normal terminal proliferation anddifferentiation. Signaling proteins activated by integrins in erythroid cells are unknown as are the identities ofgenes that are up- or down- regulated. Thus we will obtain transcriptional profiles of purified CFU-Eprogenitors cultured under conditions where integrins are adherent to fibronectin, and determine the signaltransduction pathways specifically activated. Finally we will create a framework for the transcriptionalregulatory networks active in erythropoiesis by combining expression analysis with promoter binding data todetermine which subset of Stat5 and GATA1- bound genes are actively regulated during erythroiddifferentiation, and which subset of GATA1- dependent genes require FOG. We will use a combination ofbioinformatic and experimental techniques to determine the gene(s) activated by these factors, and howmultiple transcription factors might interact to regulate erythroid- specific genes. This information will form abasis on which to establish a transcriptional regulatory network for the terminal differentiation of erythrocytesand identify key genes crucial to determining the phenotype of erythrocytes.
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