Fli-1 is required for murine vascular and megakaryocytic development and is hemizygously deleted in patients with thrombocytopenia.

The ETS gene Fli-1 is involved in the induction of erythroleukemia in mice by Friend murine leukemia virus and Ewings sarcoma in children. Mice with a targeted null mutation in the Fli-1 locus die at day 11.5 of embryogenesis with loss of vascular integrity leading to bleeding within the vascular plexus of the cerebral meninges and specific downregulation of Tek/Tie-2, the receptor for angiopoietin-1. We also show that dysmegakaryopoiesis in Fli-1 null embryos resembles that frequently seen in patients with terminal deletions of 11q (Jacobsen or Paris-Trousseau Syndrome). We map the megakaryocytic defects in 14 Jacobsen patients to a minimal region on 11q that includes the Fli-1 gene and suggest that dysmegakaryopoiesis in these patients may be caused by hemizygous loss of Fli-1.

Negative and translation termination-dependent positive control of FLI-1 protein synthesis by conserved overlapping 5' upstream open reading frames in Fli-1 mRNA.

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5' upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG -41 and GUG -37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5' uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5' uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5' uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons.

Generation of a novel Fli-1 protein by gene targeting leads to a defect in thymus development and a delay in Friend virus-induced erythroleukemia.

The proto-oncogene Fli-1 is a member of the ets family of transcription factor genes. Its activation by either chromosomal translocation or proviral insertion leads to Ewing's sarcoma in humans or erythroleukemia in mice, respectively, Fli-1 is preferentially expressed in hematopoietic and endothelial cells. This expression pattern resembled that of c-ets-1, another ets gene closely related and physically linked to Fli-1. We also generated a germ line mutation in Fli-1 by homologous recombination in embryonic stem cells. Homozygous mutant mice exhibit thymic hypocellularity which is not related to a defect in a specific subpopulation of thymocytes or to increased apoptosis, suggesting that Fli-1 is an important regulator of a prethymic T-cell progenitor. This phenotype was corrected by crossing the Fli-1 deficient mice expressing Fli-1 cDNA. Homozygous mutant mice remained susceptible to erythroleukemia induction by Friend murine leukemia virus, although the latency period was significantly increased. Surprisingly, the mutant Fli-1 allele was still a target for Friend murine leukemia virus integration, and leukemic spleens with a rearranged Fli-1 gene expressed a truncated Fli-1 protein that appears to arise from an internal translation initiation site and alternative splicing around the neo cassette used in the gene targeting. The fortuitous discovery of the mutant Fli-1 protein, revealed only as the result of the clonal expansion of leukemic cells harboring a rearranged Fli-1 gene, suggests caution in the interpretation of gene-targeting experiments that result in either no or only a subtle phenotypic alteration.

An immunological renal disease in transgenic mice that overexpress Fli-1, a member of the ets family of transcription factor genes.

The proto-oncogene Fli-1 is a member of the ets family of transcription factor genes. Its high expression in the thymus and spleen and the presence of DNA binding sites for Fli-1 in a number of lymphoid cell-specific gene suggest that Fli-1 is involved in the regulation of lymphopoiesis. Activation of the Fli-1 gene by either chromosomal translocation or viral insertion leads to Ewing's sarcoma in humans and erythroleukemia in mice, respectively. Thus, Fli-1 is normally involved in pathways involved in the regulation of cell growth and differentiation. We have generated H-2Kk-Fli-1 transgenic mice that overexpress Fli-1 in various mouse tissues, with the highest levels of Fli-1 protein in the thymus and spleen. These Fli-1 transgenic mice developed a high incidence of a progressive immunological renal disease and ultimately died of renal failure caused by tubulointerstitial nephritis and immune-complex glomerulonephritis. The incidences of renal disease correlated with the levels of Fli-1 protein in lymphoid tissues of transgenic lines. The hypergammaglobulinemia, splenomegaly, B-cell hyperplasia, accumulation of abnormal CD3+ B220+ T lymphoid cells and CD5+ B220+ B cells in peripheral lymphoid tissues, and detection of various autoantibodies in the sera of diseased Fli-1 transgenic mice suggested the involvement of an immune dysfunction in the pathogenesis of the renal disease. In addition, splenic B cells from transgenic mice exhibited increased proliferation and prolonged survival in vitro in response to mitogens. Taken together, these data suggest that overexpression or ectopic expression of Fli-1 perturbs normal lymphoid cell function and programmed cell death. Thus, H-2Kk-Fli-1 transgenic mice may serve as a murine model for autoimmune disease in humans, such as systemic lupus erythematosus.

The carbonic anhydrase II gene, a gene regulated by thyroid hormone and erythropoietin, is repressed by the v-erbA oncogene in erythrocytic cells.

The v-erbA oncogene encodes an altered form of the nuclear receptor of the thyroid hormone triiodothyronine (T3). This altered receptor is unable to bind T3, and blocks the differentiation of chicken erythrocyte progenitor cells. To identify the cellular target genes of v-ErbA in the transformed cells, we analysed the expression of several genes in normal erythrocytic cells exposed to T3, and found that the gene encoding carbonic anhydrase II is transcriptionally activated by the hormone. In contrast, this gene is repressed in erythroleukemic cells transformed by the v-erbA product. To investigate in more details the effects of v-ErbA, we constructed a mutant of v-ErbA in which we restored the ability to bind T3. This mutant developed its oncogenicity only in the absence of T3. Upon binding of T3, the transformed cells differentiated and immediately expressed the carbonic anhydrase II gene. These data show that v-ErbA directly inhibits the transcription of the carbonic anhydrase II gene, presumably by competing with normal T3 receptors. The carbonic anhydrase II gene is the first identified target gene of the v-ErbA oncoprotein in erythroleukemic cells.