Regulation of transcription of the human erythropoietin receptor gene by proteins binding to GATA–1 and Sp1 motifs

Abstract

Erythropoietin (Epo), the primary regulator of the production of erythroid cells, acts by binding to a cell surface receptor (EpoR) on erythroid progenitors. We used deletion analysis and transfection assays with reporter gene constructs to examine the transcription control elements in the 5' flanking region of the human EpoR gene. In erythroid cells most of the transcription activity was contained in a 150 bp promoter fragment with binding sites for transcription factors AP2, Sp1 and the erythroid-specific GATA-1. The 150 bp hEpoR promoter exhibited high and low activity in erythroid OCIM1 and K562 cells, respectively, reflecting the high and low levels of constitutive hEpoR expression. The GATA-1 and Sp1 binding sites in this promoter lacking a TATA sequence were necessary for a high level of transcription activation. Protein-DNA binding studies suggested that Sp1 and two other CCGCCC binding proteins from erythroid and non-erythroid cells could bind to the Sp1 binding motif. By increasing GATA-1 levels via co-transfection, we were able to transactivate the hEpoR promoter in K562 cells and non-erythroid cells, but not in the highly active OCIM1 cells, although GATA-1 mRNA levels were comparable in OCIM1 and K562. Interestingly, when we mutated the Sp1 site, resulting in a marked decrease in hEpoR promoter activity, we could restore transactivation by increasing GATA-1 levels in OCIM1 cells. These data suggest that while GATA-1 can transactivate the EpoR promoter, the level of hEpoR gene expression does not depend on GATA-1 alone. Rather, hEpoR transcription activity depends on coordination between Sp1 and GATA-1 with other cell-specific factors, including possibly other Sp1-like binding proteins, to provide high level, tissue-specific expression.

Mouse surviving solely on human erythropoietin receptor (EpoR): model of human EpoR-linked disease

“…EpoR isolated from the subject with PFCP has different functional properties than the artificially truncated mouse EpoR used by Zang et al 6 The levels of expression of the mutant EpoR were comparable to the wild-type mouse EpoR in both studies; however, in our model the level of expression was achieved only after removal of the neomycin (neo)-selectable marker by crossing the mice to cre-transgenic mice ( Figure 1). The animals reported by Zang et al 6 had the neo gene retained in the EpoR locus, albeit driven by a different promoter than that used by us.…”

“…We previously carried out careful analysis of the human EPOR proximal region and found that while much of the transcription activity is contained in a 15-base pair (bp) 5Ј proximal promoter fragment, contributions can be seen from the more distal flanking regions. 6 This is also observed in reporter gene experiments in transgenic mice for promoter fragments extending to 150-bp 5Ј and 1778-bp 5Ј (Z. Y. Liu, C. Liu, and C. T. Noguchi, unpublished data, 2002). Additional flanking sequences appear to be required to provide a high level of human EPOR expression in vivo.…”

“…The animals reported by Zang et al 6 had the neo gene retained in the EpoR locus, albeit driven by a different promoter than that used by us.…”

“…5 We have shown that mice heterozygous or homozygous for the truncated gain-of-function human EpoR were polycythemic and their erythroid progenitors had increased in vitro sensitivity to both mouse and human Epo. 2 Interestingly, another animal model of truncated EpoR without apparent polycythemic phenotype in adults and with comparable in vitro responses of wild-type and mutant erythroid progenitors to Epo was later published by Zang et al 6 The reason for this discrepancy is not clear; in both cases the resulting truncated EpoR contained one tyrosine. However, it is possible that the mutant Figure 1.…”

Epo and Non-hematopoietic Cells: What Do We Know?

“…Indeed expression of bCR in the heart and the lack of Epo effect in bCR knockout myocardium was shown (79). Whereas in hematopoietic lineage EpoR expression is induced by GATA-1, Sp1, and Wt1 transcription factors (151,152), expression of the common EpoR subunits in the heart is under control of GATA-4 and Sp1 transcription factors (145). The role of Wt1 expressed only in epicardium in regulation of EpoR expression remains to be clarified (153).…”