Lineage Specific Erythropoietin Receptor Expression Regulation

Abstract 4779

Erythropoietin receptor (EpoR) is a member of the cytokine receptor superfamily. During erythroid differentiation of hematopoietic stem cells, Epo acts through binding of EpoR on the surface of early erythroid progenitor cells to promote cell survival, proliferation and differentiation down the erythroid lineage. The extent of Epo response is dependent on the level of EpoR expression. The EpoR proximal promoter contains an inverted GATA-1 binding site (TTATCT) located at position -179 from the first codon of the human EpoR (hEpoR) gene. Three E boxes sites (CAGCTG) are also present in the 5′ untranslated transcribed region (UTR) of the EpoR gene and appear to be evolutionarily conserved in mammals. The expression of EpoR is not restricted to the erythroid lineage and can be found in several non-hematopoietic cells including endothelial, neural, muscle, cardiovascular and renal tissues. We previously found that EpoR is also expressed in primary satellite cells from skeletal muscle and in myoblast C2C12 cells but not in terminally differentiated myotubes. Epo stimulates proliferative and/or anti-apoptotic activities in myoblasts. Here we observed that erythroid MEL cells and C2C12 cells in the undifferentiated state express EpoR at a similar order of magnitude, but while EpoR expression increases with MEL cell differentiation, expression decreases with C2C12 differentiation. To gain insight on Epo activity during lineage specific differentiation, we compared EpoR expression during erythroid differentiation of MEL cells with myoblast differentiation of C2C12 cells. The conserved E-box region acts as a binding site for tissue specific basic-helix-loop-helix transcription factors, TAL1 in erythroid cells and the MyoD transcription factor family member in myoblasts. Deletion or mutation of the E-box motifs resulted in down regulated transcriptional activity of the EPO-R proximal promoter in erythroid and myoblast cells. These data suggest that the E-box region contributes to high activation of EPO-R transcription in both cell types. We also found that EPO-R expression is also dependent on the GATA-motif in the proximal promoter in myoblasts. While GATA-1, required for high level EpoR expression in erythroid cells is not expressed in myoblasts, we determined other GATA proteins in C2C12 cells associate with the GATA-1 binding site to provide transcription activity. However, unlike erythroid cells that exhibit high level induction of GATA-1 with erythroid differentiation and thus, high level EpoR expression, GATA proteins are down regulated with myoblast differentiation and EpoR expression is down regulated. Overall, during differentiation we observed an increase in histone acetylation and H3-K4 dimethylation in chromatin associated with the EpoR promoter in erythroid cells in contrast to a decrease in differentiating C2C12 cells, suggesting that the chromatin structure of EpoR in myoblast is less accessible than in erythroid cells and reflects the lower EpoR expression in non-hematopoietic cells. While DNA binding motifs for GATA and basic-helix-loop-helix transcription factors regulate EpoR expression in both erythroid and myoblast cells, important differences during lineage specific differentiation in EpoR chromatin accessibility and induction of the corresponding transcription factors that regulate EpoR expression explain in part the high induction of EpoR during erythroid differentiation in comparison to the low level expression in non-hematopoietic tissues.