Mouse models have proven invaluable for understanding erythropoiesis. Here, we describe an autosomal recessive, inherited anemia in the mouse mutant hem6. Hematologic and transplantation analyses reveal a mild, congenital, hypochromic, microcytic anemia intrinsic to the hematopoietic system that is associated with a decreased red blood cell zinc protoporphyrin to heme ratio, indicative of porphyrin insufficiency. Intercross matings show that hem6 can suppress the porphyric phenotype of mice with erythropoietic protoporphyria (EPP). Furthermore, iron uptake studies in hem6 reticulocytes demonstrate defective incorporation of iron into heme that can be partially corrected by the addition of porphyrin precursors. Gene expression and enzymatic assays indicate that erythroid 5-aminolevulinic acid synthase (Alas2) is decreased in hem6 animals, suggesting a mechanism that could account for the anemia. Overall, these data lead to the hypothesis that hem6 encodes a protein that directly or indirectly regulates the expression of Alas2.
IntroductionErythroid maturation is a complex process coordinately regulated at multiple levels. 1,2 An array of proteins act in a highly orchestrated manner to control the rapid, global changes in gene expression that occur during erythropoiesis. While the majority of genes are repressed in terminally differentiating erythroid cells, the cellular machinery involved in hemoglobin synthesis is rapidly activated. 3 Coordinated porphyrin biosynthesis, iron acquisition, and globin protein production are required to ensure that these components accumulate in the proper stoichiometry to produce the end product, hemoglobin. In general, insufficiency of porphyrin, iron, or globin impairs hemoglobin production and results in a hypochromic, microcytic anemia. 4,5 Heme, the prosthetic group of hemoglobin, is formed in a series of well-characterized catalytic steps that occur within and outside of mitochondria. The expression of all 8 heme biosynthesis enzymes is up-regulated in terminally differentiating erythroid cells, 6 and erythroid-specific promoters that include GATA1 binding sites are present in most of the heme biosynthetic genes, including 5-aminolevulinic acid synthase 2 (Alas2), which contains several erythroid-specific transcription binding sites, enhancers and DNase hypersensitivity sites in the promoter, and several introns. [5][6][7] Nonetheless, the absence of erythroid-specific promoters in the uroporphyrinogen decarboxylase (Urod) gene and the observation that GATA1 loss-of-function mutations do not reduce the protein levels of porphobilinogen deaminase (Pbgd) suggest that there are other unidentified, perhaps more universal, constituents of the porphyrin biosynthetic enzyme transcriptional regulatory machinery. 8 ALAS2 and ferrochelatase (Fech) are also regulated at the translational level, either directly by intracellular iron or indirectly through iron-sulfur clusters or heme. The 5Ј un-translated region (5ЈUTR) of the Alas2 mRNA contains an iron-responsive element (IRE), and iro...