Erythroid-specific genes contain binding sites for NF-E1 (also called GF-1 and Eryf-1; refs 1-3 respectively), the principal DNA-binding protein of the erythrocytic lineage. NF-E1 expression seems to be restricted to the erythrocytic lineage. A closely related (if not identical) protein is found in both a human megakaryocytic cell line and purified human megakaryocytes; it binds to promoter regions of two megakaryocytic-specific genes. The binding sites and partial proteolysis profile of this protein are indistinguishable from those of the erythroid protein; also, NF-E1 messenger RNA is the same size in both the megakaryocytic and erythroid cell lines. Furthermore, point mutations that abolish binding of NF-E1 result in a 70% decrease in the transcriptional activity of a megakaryocytic-specific promoter. We also find that NF-E2, another trans-acting factor of the erythrocytic lineage, is present in megakaryocytes. Transcriptional effects in both lineages might then be mediated in part by the same specific trans-acting factors. Our data strengthen the idea of a close association between the erythrocytic and the megakaryocytic lineages and could also explain the expression of markers specific to the erythrocytic and megakaryocytic lineages in most erythroblastic and megakaryoblastic permanent cell lines.

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Thrombopoietin (TPO) is the major regulator of proliferation and differentiation of megakaryocytes and their progenitors. These actions can be reproduced in the human megakaryoblastic cell line UT7 into which the murine TPO receptor, c-Mpl, was introduced. In these cells, TPO enhanced the expression of the specific megakaryocytic marker integrin glycoprotein (GP) IIbIIIa while decreasing the expression of erythroid genes (Porteu, F., Rouyez, M.-C., Cocault, L., Benit, L., Charon, M., Picard, F., Gisselbrecht, S., Souyri, M., and DusanterFourt, I. (1996) Mol. Cell. Biol. 16, 2473-2482). We have now analyzed the effect of TPO on the transcriptional activity of the GPIIb promoter in these cells. Using transient transfection assays of a series of human GPIIb promoter fragments, we delineated a TPO-responsive element within the previously reported enhancer region of the promoter. Although this enhancer included GATA-and Ets-binding sites (EBSs), we found that only EBS ؊514 was important for TPO response. We identified PU.1/Spi-1 as the endogenous Ets transcription factor that strongly and preferentially interacted with this enhancer EBS. This factor did not interact with other proximal EBSs in the GPIIb promoter. We next showed that TPO induced a strong and selective increase of PU.1/Spi-1 expression and DNA binding activity in UT7-Mpl cells. In contrast, TPO did not affect the expression of Ets-1/2 while weakly increasing the levels of Fli-1. Overexpression of PU.1/Spi-1 was further shown to enhance GPIIb promoter activity in the absence and presence of TPO. Overall, our data indicated that, in UT7-Mpl cells, TPO increased the transcriptional activity of a GPIIb gene in part due to an enhanced expression of an unexpected transcription factor, the Ets family PU.1/ Spi-1 factor. To our knowledge, this is the first evidence of a role for the PU.1/Spi-1 factor in the regulation of megakaryocytic genes.Megakaryocytic differentiation is characterized by the increase of DNA content in the cell, which involves an endomitotic process. This will lead to an increase in megakaryocyte ploidy and size and to the ultimate production of platelets from the fragmentation of megakaryocyte cytoplasm. Megakaryocytic differentiation is also characterized by the synthesis of a number of platelet proteins. Among these, the integrin ␣ IIb ⅐␤ 3 complex (glycoprotein (GP) 1 IIb-IIIa); the glycoproteins GPIb, GPIX, and GPV; platelet factor 4; and the ␤-thromboglobulin are exclusively expressed on platelets and megakaryocytes. In the past few years, the 5Ј-regulatory regions of a number of megakaryocytic genes were cloned and sequenced (1-7). These promoter regions were shown to share many characteristic features. In particular, they all contain associated binding sites for GATA and Ets factors (2,5,8,9). The ␣ IIb (GPIIb) gene is one of the most studied megakaryocytic genes. This TATA-less gene encodes a protein, the ␣-subunit of integrin ␣ IIb ⅐␤ 3 , which is expressed at a very early stage of megakaryocytic differentiation. We (10, 11) and o...

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder, caused by a quantitative or qualitative defect of the GPIIb-IIIa integrin (alpha IIb beta 3), which functions as the platelet fibrinogen receptor. We report a case of type I GT due to a homozygous mutation resulting in Ser 870 to stop codon substitution. This residue is located near the proteolytic cleavage site of proGPIIb. The mutation results in a GPIIb truncated of 138 amino acids, including transmembrane and intracytoplasmic domains. Cotransfection of an expression vector containing the mutant GPIIb and wild-type GPIIIa showed that the mutant Ser 870-->stop GPIIb was able to associate to GPIIIa. However, this heterodimer failed to mature as shown by endoglycosidase-H digestion and was therefore not expressed at the COS-7 cell surface. This report is the first description of a homozygous nonsense mutation in the GPIIb gene and highlights the role of the GPIIb light chain.