Retroviral insertions that activate proto-oncogenes are a primary cause of tumors in certain strains of mice. The AKXD recombinant inbred mice are predisposed to a variety of leukemias and lymphomas as a result of viral integration. One common insertion site, the ecotropic viral insertion site 3 (Evi3), has been implicated in most B-cell tumors in the AKXD-27 strain. The Evi3 gene encodes a zinc-finger protein with sequence similarity to the Early B-cell Factor-Associated Zinc-finger gene (EBFAZ). We show that the Evi3 gene is overexpressed in several tumors with viral insertions at Evi3, which results in the upregulation of Early B-cell Factor (EBF)-target gene expression, suggesting that Evi3 modulates EBF activity. Reconstitution of primary leukemia cells showed that these tumors express high densities of the Bcell surface proteins CD19 and CD38, which are EBF targets. Using a transactivation assay, we show that the terminal six zinc-fingers of Evi3 are required for modification of EBF activity. This is the first evidence that Evi3 expression in tumors alters the level of EBF target genes, and the first characterization of the Evi3 protein domains required for modulation of EBF activity. Further, these data imply that Evi3 misexpression initiates tumorigenesis by perturbing B-cell development via an interaction with EBF.

Mutations in the melanocortin-4 receptor (MC4R) are associated with early-onset obesity in humans. Furthermore, a null Mc4r allele in mice leads to severe obesity due to hyperphagia and decreased energy expenditure. As part of independent N-ethyl- N-nitrosourea (ENU) mutagenesis screens, two obesity mutants, Fatboy and Southbeach, were isolated. Mapping revealed linkage to the melanocortin-4 receptor (Mc4r) and sequencing found single amino acid changes in Mc4r for each line. Expression of the mutant receptors in HEK 293 cells revealed defects in receptor signaling. The mutated Fatboy receptor (I194T) shows an increase in the effective concentration necessary for 50% of maximal signaling (EC(50)) when stimulated with alpha-MSH. Based on competitive binding, I194T is expressed on the cell surface at lower levels than the nonmutated receptor. In contrast, Southbeach (L300P) displays minimal receptor signaling when stimulated with the natural ligand alpha-MSH or the synthetic agonist NDP-alpha-MSH. Cell surface binding is absent, which usually indicates a lack of cell surface expression. However, antibody binding to Flag-tagged receptors by flow cytometry analysis and immunofluorescence demonstrates that L300P is translocated to the plasma membrane at a level comparable to the wild-type receptor. These results indicate a correlation with remaining receptor activity and the severity of the obesity in the mice homozygous for the mutations. Southbeach has less receptor activity and becomes more obese. These mutants will serve as good models for the variability in phenotype in humans carrying mutations in the MC4R gene.

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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...

Mouse mutagenesis using forward genetics is valuable as a gene function discovery tool. We are looking for blood defects in a large ENU mutagenesis screen, and have isolated many new mouse mutants that reveal new mechanisms in hematopoiesis. One mutant mouse strain, called redears, is an intriguing model of inflammatory disease and thrombocytopenia. Animals homozygous for the redears (rd) mutation develop spontaneous inflammatory lesions of the ears and tail characterized by neutrophil infiltration and peripheral neutrophila. Unexpectedly, blood platelet numbers are dramatically reduced in rd/rd animals. A thorough analysis of platelet biogenesis shows that the platelet precursor cell, the megakaryocyte, undergoes abnormal maturation, which results in gross morphological abnormalities, increased ploidy and abortive platelet shedding. Here we report a mutation in a novel gene related to the yeast actin-interacting protein Aip1 in rd/rd mice. In yeast, Aip1 interacts with, and increases the activity of cofilin, a key regulator of actin depolymerization. Our data confirm that actin dynamics are dysregulated in rd/rd megakaryocytes and neutrophils. The massive cytoplasmic reorganization that is required for megakaryocyte maturation and platelet shedding has long been assumed to depend on the actin cytoskeleton. Intriguingly, recent studies suggest the process is caspase-dependent, and represents a form of ‘para-apoptosis’. With this in mind, we found that chemotaxis and apoptosis are perturbed in rd/rd neutrophils, suggesting that neutrophils are playing a key role in driving the inflammation. Disrupted actin depolymerization would provide an explanation for chemotactic deficiencies. Further, recent evidence implicating cofilin and other actin regulators in the initiation of apoptosis would suggest that this novel protein may play an essential role in neutrophil cell death. Thus, the redears mouse not only provides the first in vivo demonstration of the critical role of the actin cytoskeleton in megakaryocyte development and platelet production, but also represents a unique reagent to examine the relationship between actin dynamics, cellular maturation, inflammation and apoptosis. Our ongoing mutagenesis efforts continue to reveal new developmental mechanisms. New mutants, genetic tools, and resources can be found at www.mouse-genome.bcm.tmc.edu