Magness S T, Tugores A, Brenner D A
Departments of Medicine and Biochemistry and Biophysics, Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7038, USA.
Blood. 2000 Jun 1;95(11):3568-77.
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 cognate trans-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.
亚铁螯合酶是血红素合成途径中的最后一种酶，它将原卟啉IX与铁螯合形成血红素，在原卟啉症中发生突变。亚铁螯合酶基因在所有组织中低水平表达，为必需的含血红素蛋白提供血红素，并在红细胞生成过程中上调以合成血红蛋白。人类亚铁螯合酶启动子包含2个Sp1顺式元件以及GATA和NF-E2位点，所有这些元件在体外均能结合其相应的反式作用因子。为了研究这些元件在红细胞生成过程中的作用，我们将由各种亚铁螯合酶启动子片段驱动的绿色荧光蛋白（EGFP）转基因表达导入小鼠胚胎干细胞的单个位点。使用流式细胞术在体外造血分化过程中监测EGFP表达。我们发现，一个包含Sp1位点、NF-E2和GATA元件的启动子片段足以赋予EGFP转基因发育特异性表达，其表达谱与内源基因相同。在这个系统中，-0.275 kb的NF-E2顺式元件是红细胞增强表达所必需的，GATA顺式元件作为阶段特异性阻遏物和增强子发挥作用，位于-0.375 kb和-1.1 kb之间的元件对于最佳表达水平是必需的。亚铁螯合酶mRNA在原始红细胞阶段之前增加，但亚铁螯合酶蛋白没有相应增加，这表明存在翻译控制机制。由于该系统的敏感性，我们能够评估在原卟啉症患者启动子中鉴定出的A到G多态性的影响。G单倍型对-1.1 kb转基因的转录活性没有影响。
