Analysis of ferrochelatase expression during hematopoietic development of embryonic stem cells

Abstract

Ferrochelatase, the last enzyme in the heme pathway, chelates protoporphyrin IX and iron to form heme and is mutated in protoporphyria. The ferrochelatase gene is expressed in all tissues at low levels to provide heme for essential heme-containing proteins and is up-regulated during erythropoiesis for the synthesis of hemoglobin. The human ferrochelatase promoter contains 2 Sp1 cis-elements and GATA and NF–E2 sites, all of which bind their cognatetrans-acting factors in vitro. To investigate the role of these elements during erythropoiesis, we introduced expression of the green fluorescent protein (EGFP) transgenes driven by various ferrochelatase promoter fragments into a single locus in mouse embryonic stem cells. EGFP expression was monitored during hematopoietic differentiation in vitro using flow cytometry. We show that a promoter fragment containing the Sp1 sites, the NF–E2 and GATA elements, was sufficient to confer developmental-specific expression of the EGFP transgene, with an expression profile identical to that of the endogenous gene. In this system the −0.275 kb NF–E2 cis-element is required for erythroid-enhanced expression, the GATA cis-element functions as a stage-specific repressor and enhancer, and elements located between −0.375kb and −1.1kb are necessary for optimal levels of expression. Ferrochelatase mRNA increased before the primitive erythroid-cell stage without a concomitant increase in ferrochelatase protein, suggesting the presence of a translational control mechanism. Because of the sensitivity of this system, we were able to assess the effect of an A-to-G polymorphism identified in the promoters of patients with protoporphyria. There was no effect of the G haplotype on transcriptional activity of the −1.1 kb transgene.