To search for new genes that might be involved in the regulation of erythropoiesis, we performed cDNA representational difference analysis on a multipotent murine hematopoietic cell line, EML C1. Penumbra was identified based on its predominant, high-level expression in TER119+ erythroblasts. It is not expressed or is expressed at very low levels in Gr1+ or CD3+ or B220+ or CD11c or NK1.1+ bone marrow or spleen cells. The full-length murine Penumbra mRNA contains 2017 nucleotides and encodes a new membrane protein characterized by four transmembrane domains, a small extracellular domain between the first and second transmembrane domains and a large extracellular domain between the third and fourth transmembrane domains. It is a member of the evolutionarily conserved tetraspanin membrane protein superfamily that includes CD63, CD81, CD151 and Peripherin. Using the murine Penumbra as a probe, we identified the human Penumbra from a human bone marrow cDNA library. The human Penumbra also encodes a protein of 283 amino acids and is 97% identical to the murine Penumbra. The human Penumbra gene is mapped to chromosome 7q32, a hotspot for interstitial deletions in myelodysplastic syndromes and acute myelogenous leukemias. Immunofluorescence microscopy indicated that Penumbra is targeted to the cell surface. Immunoprecipitation-Western analysis showed that Penumbra formed disulfide bond-linked homodimers. To study the possible effects of Penumbra deletions, a knockout mouse model was created by homologous recombination. To avoid any pathology that might result from the Neo selectable marker, the targeting vector employed a self-excising Cre-Neo cassette driven by a testis-specific promoter. At 3 months of age, Penumbra/− mice had similar numbers of RBC, hematocrits, WBC and platelets as the WT littermates. At 1 year of age, Penumbra/− mice as a group had significantly lower RBC numbers and hematocrits than the WT littermates. Furthermore, about 40–60% of the Penumbra/− mice had increased percentages of basophilic or polychromatophilic macrocytic RBC. As a group, these mice had even lower numbers of RBC (6.24 ± 1.87 vs. 8.88 ± 0.51 x 106/microliter in WT; p = 0.0002) and hematocrits (36.63 ± 8.28 vs. 46.21 ± 3.62% in WT; p = 0.002). Many such mice had massive splenomegaly due to increased extramedullary hematopoiesis. A multipotent hematopoietic cell line, EMX, was established from the bone marrow of a Penumbra/- mouse. EMX exhibits ineffective erythropoiesis in response to erythropoietin, a defect that is reversed by re-expression of Penumbra. These findings indicate that Penumbra plays an important role in erythropoiesis and suggest that Penumbra may be one of the genes that are often deleted during the evolution of myelodysplastic syndromes and acute myelogenous leukemias.

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