Regulation of the erythropoietin gene
Erythropoietin (Epo) is a circulating hormone which regulates red blood cell production, and hence oxygen-carrying capacity. Three striking features of Epo gene expression are that it shows high level induction in response to hypoxia, that it is tightly tissue-restricted and that it is deficient in most forms of renal damage. Each of these aspects has been investigated in this thesis. Chapter 3 concerns the hypoxic-sensing system which induces Epo gene expression via an enhancer element in cultured hepatoma cells. Transient transfection was used to examine a range of cell-lines, which did not produce Epo, for the presence of this oxygen-sensing system. It was demonstrated that such a system is widespread in mammalian cells. It is probable that it is involved in regulating the expression of other genes in response to hypoxia. This widespread oxygen-sensing mechanism contrasts with the tightly tissue-restricted expression of the Epo gene. Transgenic experiments described in Chapters 4 and 5 provide data concerning the DNA sequences involved in this tissue specificity. Sequence from the mouse Epo locus was then used to direct expression of a marker protein, SV40 T antigen, to Epo-expressing cells. This led to identification of the Epo-producing cells in the kidney (Chapter 5) and liver (Chapter 6). In both organs a fibroblast-like population expresses the gene; the Ito cells in the liver and the Type 1 interstitial cells in the kidney. In the liver hepatocytes also produce Epo. Further studies of Epo gene expression would be facilitated by the availability of Epo-producing cell lines. SV40 T antigen is a viral oncogene, and expression of this protein was used as a proliferative stimulus in vivo. The effects of this, and attempts to isolate these cells form the subject of Chapter 7. Chapter 7 also describes experiments in which renal injury was combined with visualisation of individual Epo-producing cells. A homologous recombination at the Epo locus resulted in an allele which expressed the hormone at a greatly reduced level. The phenotype of animals homozygous for this allele is described in Chapter 8. Experiments designed to produce further modification of the Epo locus by this method, and to assess its utility in modifying the mouse genome are also described.