Different domains regulate the human erythropoietin receptor gene transcription

Abstract

To analyse the 5'-flanking sequences required for the tissue specific transcription of the human erythropoietin receptor (hEpo-R) gene, a DNA region spanning nucleotides -1050 to +135 relative to the transcription initiation site (+1) was explored. Our studies indicate that a minimum promoter (-76/+33) containing GATA and SP1 binding sites at positions -45 and -20 is not sufficient to confer erythroid specific expression to a reporter gene. Erythroid specificity of the promoter was observed either with the (-1050/+33 construct) which contains a cluster of Alu repetitive elements or with the addition of the 135 bp down to the transcription initiation site (-76/+135 construct) which exert a negative control on the promoter activity with a major effect in non erythroid tissues. The latter region can be subdivided on two distinct domains: the +1/+78 region that exerts a positive effect and the +79/+135 region that has a negative effect on the Epo-R promoter activity measured by CAT assays. The first region contains three CANNTG motifs, whereas the second contains an SP1/CACCC motif at position +85. These findings reveal a complex regulation of the hEpo-R gene and provide a working model useful to explain how the minimal promoter, containing GATA/SP1, can be positively and negatively regulated during erythroid differentiation.

The human erythropoietin receptor gene rescues erythropoiesis and developmental defects in the erythropoietin receptor null mouse

“…4 This transgene includes 6 kb of 5Ј flanking sequence containing the proximal promoter that provides much of the transcription activity in erythroid cells. [5][6][7] We show that the hEpoR transgene restores effective erythropoiesis in the EpoR null mouse and normalizes cardiac as well as brain development. These results illustrate that the mouse and human EpoRs are nearly equivalent and provide evidence for erythropoietin multiorgan function.…”

Transcriptional Regulation of Neuronal Nicotinic Acetylcholine Receptor Genes

“…In this report we demonstrated conclusively that the transcriptional activator Sp1 associates with a CA box in the 5Ј-flanking region of the rat ␤4 subunit gene and can activate ␤4 promoter/luciferase constructs in transient transfection experiments. The CA box has been demonstrated to bind distinct proteins in a variety of cell types and participates in the regulation of such genes as the T-cell receptor (44), the erythropoietin receptor (45), members of the globin gene family (47)(48)(49), the glucocorticoid receptor (46), and others (52)(53)(54). Indeed, a mutation within the CA box of the ␥-globin gene promoter has been shown to result in the development of ␤-thalassemia (56 -57).…”

Phosphatidylinositol 3-Kinase/Akt Induced by Erythropoietin Renders the Erythroid Differentiation Factor GATA-1 Competent for TIMP-1 Gene Transactivation

“…Transient transfection was carried out in 10-cm dishes, and the total amount of DNA in each transfection was kept constant at 5 g by adding the pUC19 plasmid as backbone. Lipofectamine (Gibco BRL) was used as previously described for COS-7 cells (20). For UT7 or FDC-P1/Epo-R, cells were washed with Iscove's modified Dulbecco's medium, seeded at densities of 4 ϫ 10 6 and 10 ϫ 10 6 cells/well, respectively, in six-well plates and transfected with Lipofectamine according to manufacturer's instructions.…”