Erythropoietin Receptor Expression: A Role for the Tal1/SCL and GATA-1 Complex

Tal1/SCL is required for hematopoietic stem cell development and is essential for normal erythropoiesis. Although Tal1-DNA binding is not required for hematopoietic development, Tal1 participates in a complex with other binding partners GATA-1, LMO2, Ldb1 and possibly Sp1. During erythropoiesis, Tal1 can activate expression via direct association with E-box-GATA DNA sequence motifs for lineage specific genes such as Protein 4.2. In mice, loss of Tal1 in adult erythropoiesis also affects TER119 expression and BFU-E growth resulting in anemia. We found that knock down of Tal1 expression in primary erythropoietin (EPO) stimulated hematopoietic progenitor cells in culture inhibited EPO receptor (EPOR) expression and erythroid differentiation, consistent with the anemia observed in mice with targeted deletion of adult Tal1 expression. In contrast, overexpression of Tal1 in erythroid cells increased both EPOR expression and erythroid differentiation. In fact, overexpression of EPOR was sufficient to increase differentiation in erythroid progenitor cell cultures. Increased EPOR expression by Tal1 was mediated by 3 conserved E-boxes in the 5′ UTR. In reporter gene assays in K562 erythroid cells, EPOR promoter activity was lost with the mutation of these E-boxes, which are located 75 bp downstream of the required GATA-1 binding motif in the human EPOR proximal promoter. Tal1/E2A dimer binding to the E-box region was demonstrated by gel mobility shift assay. Tal1 transactivated EPOR mRNA and chromatin immunoprecipitation (ChIP) analysis confirmed that Tal1 bound directly to the E-box region in intact erythroid cells. We previously showed that GATA-1 also transactivates EPOR gene expression and is required for high level of EPOR transcription activity. In ChIP assays, an antibody to Tal1 also pulled down chromatin containing the GATA-1 binding site as well as the E-box region and conversely, antibodies to GATA-1 pulled down chromatin corresponding to the GATA-1 binding site and the E-box region. These data show that the complex containing Tal1 and GATA-1 bound to the E-box region and to the GATA-1 site in the EPOR proximal promoter and that the complex has greater occupancy at the GATA-1 site in the proximal promoter. High-level Tal1 expression increased chromatin containing Tal1 or GATA-1 associated with the GATA-1 binding site as well as the down stream E-box region, with a greater proportionate increase in the E-box region. Hence, in addition to the GATA-1 binding site, the downstream E-box region is necessary for high-level EPOR expression. These data suggest that transactivation of the EPOR promoter via these sites is mediated by a Tal1 and GATA-1 containing complex and that during erythropoiesis EPO induction of both Tal1 and GATA-1 regulates the high expression of its own receptor. We hypothesize that the high binding of GATA-1 and Tal1 at the GATA site opens the upstream chromatin structure. The over-expression of Tal1 promotes the open status and facilitates the spreading of open chromatin along the chromatin to the downstream E-box region. Subsequent E-box binding by Tal1 and its interaction with GATA-1 maintains the open chromatin and activates transcription.