A CCACC motif mediates negative transcriptional regulation of the human erythropoietin receptor.

We have previously shown that the +79 to +135 fragment of the human erythropoietin receptor (Epo-R) acts negatively on the transcriptional activity and confers erythroid specificity to the gene [Maouche, L., Cartron, J.-P. & Chretien, S. (1994) Nucleic Acids Res. 22, 338-346]. In this work, we demonstrate that this effect is mediated by a CCACC motif that binds weakly to the simian virus 40 protein 1 (Sp1) factor and that the increase of the affinity for Spl augments transcription inhibition. The repression is not restricted to the human Epo-R promoter, although it seems more efficient on heterologous promoters of erythroid genes. In chloramphenicol acetyl transferase constructs containing the mouse Epo-R promoter, rearranged by retroviral long terminal repeat (LTR) insertion of murine erythroleukemia cell lines, we found that positioning the CCACC motif 3' to the LTR represses the transcriptional activity mediated by the LTR in non-erythroid cells. These results demonstrate that Epo-R gene expression is negatively regulated by a CCACC or a GC box-binding factor, which is most likely identical to the Sp1 transcription protein. Further data suggest that Sp1-mediated negative regulation is not the result of a direct competition between Spl and another DNA-binding protein.