Epo, Integrins and the Control of Erythrpoiesis

Boston Children'S Hospital, Boston MA

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Abstract

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