Analysis of ferrochelatase gene regulation in transgenic mice

Abstract

Ferrochelatase, the terminal enzyme in the heme biosynthetic pathway, chelates ferrous iron and protoporphyrin IX to form heme. The inherited disease protoporphyria is caused by mutations in the human ferrochelatase (MFC) gene. Our previous studies have demonstrated that transcription of the MFC gene is regulated in cultured cells by both erythroid specific factors NFE2 and GATA-1, and constitutive SpMike factors, to deliniate the role of cis-acting elements in. the ferrochelatase gene in vivo, we have constructed two sets of transgenic mouse lines which express the luciferase (Luc) reporter gene driven by either the minimal MFC promoter (-150 MFC), devoid of the erythroid -specific elements, or with the erythroid-specific GATA-1 and NF-E2 elements present (-375 MFC). A hemotytic anemia was induced in some transgenic mice by Nacetyl-phenylhydrazine (PHZ) administered i.p. for seven days which produces extramedullary erythropoiesis. Luciferase activity in the liver and spleen was measured and compared to control transgenic mice. The Luc activity (units/mg protein) for liver and spleen in control animals was an average of 3788 and 1813, respectively, (for -ISO MFC mice) compared with 203 and 48873, respectively, (for -375 mice). Upon treatment with PHZ, luciferase activity increased in the liver and spleen an average of 3.7- and 10- fold, respectively, (for -150 MFC mice) and 9- and 10-fold, respectively, (for -375 MFC mice). We also compared the induction of the endogenous FC protein end FC activity in PHZ treated and control animals. Upon PHZ induction, the FC protein levels were unchanged in the liver and induced 4-fold in the spleen. In contrast there was a 12- to 16- fold increase in FC activity in the liver and spleen respectively. In conclusion, the HFC gene contains discrete domains that are inducible in vivo, but maximal transcriptional activity requires the erythroid-specific GATA-1 and NF-E2 binding sites. The induction of transcriptional activity exceeds protein levels and catalytic activity probably as a result of the dynamics of extramedullary erythropoiesis.