Epo, Integrins and the Control of Erythrpoiesis

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Erythropoietin (Epo) activates multiple intracellular signal transduction pathways, including StatS, PI -3 kinase/Akt, and Shc/Ras/ MARK, that interact with key erythroid transcription factors including GATA1 and FOG to prevent apoptosis of CFU-E progenitors, trigger 3-5 terminal cell divisions, and induce multiple genes essential for erythrocyte formation. Most erythroid- specific genes induced by StatS, GATA1, and FOG are unknown. Here we will use a combination of chromatin immunoprecipitation with mouse promoter microarrays (ChlP-on-chip) to identify promoters bound by Stat5b, GATA1, and FOG in erythroid progenitors and subsequent stages of erythroid differentiation purified from mouse fetal livers. In parallel we will determine the mRNA expression profiles of erythroid progenitors at different stages of terminal erythroid differentiation 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 will examine the transcriptional profiles in primary erythroid cells deficient in EpoR- mediated Akt activation or overexpressing a constitutively active Akt kinase. Recently we showed that erythroid progenitors must adhere to fibronectin via alpha4beta1, or alpha5beta1 integrin in order to undergo normal terminal proliferation and differentiation. Signaling proteins activated by integrins in erythroid cells are unknown as are the identities of genes that are up- or down- regulated. Thus we will obtain transcriptional profiles of purified CFU-E progenitors cultured under conditions where integrins are adherent to fibronectin, and determine the signal transduction pathways specifically activated. Finally we will create a framework for the transcriptional regulatory networks active in erythropoiesis by combining expression analysis with promoter binding data to determine which subset of Stat5 and GATA1- bound genes are actively regulated during erythroid differentiation, and which subset of GATA1- dependent genes require FOG. We will use a combination of bioinformatic and experimental techniques to determine the gene(s) activated by these factors, and how multiple transcription factors might interact to regulate erythroid- specific genes. This information will form a basis on which to establish a transcriptional regulatory network for the terminal differentiation of erythrocytes and identify key genes crucial to determining the phenotype of erythrocytes.