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J. Biol. Chem., Vol. 265, Issue 24, 14100-14104, Aug, 1990

Evidence suggesting negative regulation of the erythropoietin gene by ribonucleoprotein

N Beru, D Smith and E Goldwasser
Department of Medicine, University of Chicago, Illinois 60637.

The promoter regions of the mouse and human erythropoietin genes have regions of identity within 130 base pairs upstream of the cap site, suggesting a cis-acting regulatory role for the conserved sequences. We have used a double-stranded deoxyoligonucleotide corresponding to the - 61 to -45 region relative to the start site of transcription of the mouse gene in DNA mobility shift assays. Nuclear extracts from kidneys of both control and cobalt-stimulated mice contain factors that bind to this oligonucleotide in a specific manner. One factor is a 47-kDa protein, whereas the others may be one or more ribonucleoproteins. Under denaturing conditions, four RNA species which show specific binding to the oligonucleotide were observed, suggesting that recognition of the oligonucleotide by ribonucleoprotein is mediated by the RNA component. In nuclear extracts of kidneys from stimulated animals, the amount of the two largest RNA species that bind to the oligonucleotide was reduced relative to that of control, whereas the other RNA species as well as the 47-kDa protein remained relatively unaffected. These results suggest that the ribonucleoprotein containing the down-regulated RNA species may be a negative transcriptional factor and that activation of the erythropoietin gene by cobalt salts may involve, in part, decreased binding of this factor, thus allowing transcription to proceed.
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