Biomedical subjects

G Uzan

Publications and source records attributed to G Uzan.

At least 19 recordsLinked to original sources

PubMed2006

Fate and characterization of circulating tumor cells in a NOD/SCID mouse model of human hepatocellular carcinoma.

There is much debate about the way in which epithelial tumors metastasize. It has been proposed that the bone marrow (BM) acts as a tumor cell reservoir. We injected human hepatocellular carcinoma (HCC) cells (Mahlavu cell line) into the livers, circulation or BM of NOD/SCID mice and circulating tumor cells were quantified. When injected under the Glisson capsule, a primary tumor developed and continuously yielded circulating tumor cells. Liver tumor removal led to a very low level of Mahlavu cells both in blood and BM 30 days later. When Mahlavu cells (cultured or from BM of primary mice femurs) were intravenously injected into mice, the number of cells in the bloodstream (BS) steadily decreased, whereas the BM was not significantly colonized. When Mahlavu cells were directly injected into one femur, the controlateral femur was not colonized. Microscopic analysis and a sensitive PCR assay (<1 Mahlavu cell/nuclear cells) both failed to detect human tumor cells in other organs regardless of injection route. In conclusion, our model strongly supports the hypothesis that HCCs continuously release cells into the BS. However, in sharp contrast with the current hypothesis, the BM is not specifically colonized by tumor cells but could store them at a very low level.

Animals↗

PubMed2004

Probing platelet factor 4 alpha-granule targeting.

The storage mechanism of endogenous secretory proteins in megakaryocyte alpha-granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well-known platelet alpha-granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4-GFP), were transfected in the well-characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno-electronmicroscopy. In preliminary experiments, GFP, a non-secretory protein (no signal peptide), localized in the cytoplasm, while PF4-GFP colocalized with adrenocorticotropin hormone (ACTH)-containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4-GFP localized in alpha-granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify alpha-granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4-GFP), GFP was not stored into granules in spite of its efficient translocation to the ER-Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in alpha-granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4-GFP appears as an appropriate probe with which to analyze alpha-granule biogenesis and its alterations in the congenital defect gray platelet syndrome.

Adrenocorticotropic Hormone↗

PubMed2002

Expression of coagulation factor IX in a haematopoietic cell line.

We have developed a gene therapy project for haemophilia B which aims to express factor IX (FIX) in haematopoietic lineage. Haematopoietic stem cells and subsequent megakaryocyte-derived cells represent the target cells of this approach. Our speculation is that platelets can deliver the coagulation factor at the site of injury, and subsequently correct the haemostasis defect. In order to direct FIX expression in cells from the megakaryocytic lineage, we designed a FIX cassette where the FIX cDNA was placed under the control of the tissue-specific glycoprotein IIb (GPIIb) promoter. In stably transfected HEL cells, FIX production was higher when driven by the GPIIb promoter compared to the CMV promoter. Using a cassette containing both the GPIIb promoter and a truncated FIX intron 1, FIX synthesis was dramatically increased in HEL cells. Northern blot analysis demonstrated an increase in FIX mRNA amounts, which paralleled with an increase of FIX antigen in the culture supernatants. Using a one-stage clotting assay and an activation by FXIa and FVIIa/TF, the HEL-derived recombinant FIX was shown to be a biologically active protein. This recombinant protein exhibited a 60-kDa molecular mass and was more heterogeneous than plasma immunopurified FIX (Mononine). The molecular mass difference could be partly explained by a different glycosylation pattern. The GPIIb promoter appears therefore to be a very attractive sequence to specifically direct FIX production in the megakaryocytic compartment of hematopoietic cells. These data also demonstrate that hematopoietic cells may represent potential target cells in an approach to gene therapy of haemophilia B.

Factor IX↗

PubMed2000

The alpha(IIb)beta(3) integrin and GPIb-V-IX complex identify distinct stages in the maturation of CD34(+) cord blood cells to megakaryocytes.

Megakaryocytopoiesis is a complex multistep process involving cell division, endoreplication, and maturation and resulting in the release of platelets into the blood circulation. Megakaryocytes (MK) progressively express lineage-restricted proteins, some of which play essential roles in platelet physiology. Glycoprotein (GP)Ib-V-IX (CD42) and GPIIb (CD41) are examples of MK-specific proteins having receptor properties essential for platelet adhesion and aggregation. This study defined the progressive expression of the GPIb-V-IX complex during in vitro MK maturation and compared it to that of GPIIb, an early MK marker. Human cord blood CD34(+) progenitor cells were cultured in the presence of cytokines inducing megakaryocytic differentiation. GPIb-V-IX expression appeared at day 3 of culture and was strictly dependent on MK cytokine induction, whereas GPIIb was already present in immature CD34(+) cells. Analysis by flow cytometry and of the messenger RNA level both showed that GPV appeared 1 day later than GPIb-IX. Microscopy studies confirmed the late appearance of GPV, which was principally localized in the cytoplasm when GPIb-IX was found on the cell surface, suggesting a delayed program of GPV synthesis and trafficking. Cell sorting studies revealed that the CD41(+)GPV(+) population contained 4N and 8N cells at day 7, and was less effective than CD41(+)GPV(-) cells in generating burst-forming units of erythrocytes or MK colonies. This study shows that the subunits of the GPIb-V-IX complex represent unique surface markers of MK maturation. The genes coding for GPIb-IX and GPV are useful tools to study megakaryocytopoiesis and for tissue-specific or conditional expression in mature MK and platelets. (Blood. 2000;96:4169-4177)

Antigens, CD34↗

PubMed2000

Identification of a GATA-overlapping sequence within the enhancer of the murine GPIIb promoter that induces transcriptional deregulation in human K562 cells.

The human and the murine glycoprotein platelet IIb (GPIIb) promoters are megakaryocyte specific in human and murine cell systems, respectively. Here we show that the murine promoter is, however, highly active when transfected in K562 human cells in which the human promoter is almost inactive. A murine promoter, in which the enhancer element was replaced by the human, retrieves its megakaryocytic specificity in human cell lines. The human and murine GATA-binding sites located in the enhancer region display slight sequence divergence next to the consensus GATA core sequence. Gel shift experiments show that, although the murine and the human GATA sequences both bind GATA-1, the murine sequence alone forms an additional complex (B) not detected with the human sequence. When the murine GATA-containing region is replaced by the human in the context of the murine GPIIb promoter, megakaryocyte specificity is restored in the human cell lines. A G nucleotide 3 to GATA appears crucial because its substitution abrogates B but not GATA-1 binding and restores megakaryocyte specificity to the murine promoter. Conversely, substitution of the human GATA-1 binding sequence by its murine homologue that binds both GATA-1 and complex B induces an abnormal activity for the human promoter in K562 cells. Altogether, our data suggest that limited changes in the GATA-containing enhancer of the GPIIb promoter can induce the recruitment of accessory proteins that could be involved in alteration of a megakaryocyte-restricted gene activation program. (Blood. 2000;96:1348-1357)

Animals↗

PubMed1999

Functional characterization of the human platelet glycoprotein V gene promoter: A specific marker of late megakaryocytic differentiation.

Glycoprotein V (GPV), a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin, is specifically found in platelets and mature megakaryocytes. Studies of the GPV gene can therefore provide insight into the mechanisms governing megakaryocyte differentiation. The human GPV promoter was isolated, and elements important for its tissue specific transcriptional activity were localized using systematic DNase I protection and reporter deletion assays. A -1413/+25 fragment inserted into a luciferase reporter construct displayed promoter activity in Dami and HEL but not in K562, HL60, or HeLa cells. Progressive 5' to 3' deletion showed a putative enhancer region in the -1413/-903 segment that contained closely spaced GATA and Ets sites protected from DNase I digestion in Dami extracts. Regions similar to a GPIIb gene repressor were found at -816 and -610, with the first exhibiting repressor activity in Dami and HEL cells and the second protected from DNAse I. Deletions from -362 to -103, an area containing protected sites for Sp1, STAT, and GATA, induced a progressive decrease in activity. The -103/+1 fragment, bearing a proximal Ets footprinted site and a GATA/Ets tandem footprint, displayed 75% activity relative to the full-length promoter and retained cell specificity. In summary, this work defines several regions of the GPV gene promoter important for its activity. It contains megakaryocyte-specific signals, including erythro-megakaryocytic GATA, and Ets cis-acting elements, GPIIb-like repressor domains, and binding sites for ubiquitous factors such as Sp1, ETF, and STAT.

Base Sequence↗

PubMed1997

Thrombopoietin-induced expression of the glycoprotein IIb gene involves the transcription factor PU.1/Spi-1 in UT7-Mpl cells.

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) IIb-IIIa 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 Dusanter-Fourt, 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.

Binding Sites↗

PubMed1997

The MS-5 murine stromal cell line and hematopoietic growth factors synergize to support the megakaryocytic differentiation of embryonic stem cells.

Murine embryonic stem (ES) cells are able to differentiate into erythroid, mast, and granulomonocytic cells by using appropriate culture conditions. Because we were interested in the regulation of tissue-specific expression of the platelet glycoprotein IIb gene, we studied the culture conditions, aiming at the reproducible production of myeloid cells that included megakaryocytes (MKs) from ES cells. We showed that even a complex cocktail of HGFs (stem cell factor, interleukin 3, IL6, IL11, granulocyte colony-stimulating factor, and erythropoietin) is unable to induce significant myeloid differentiation in day 12 embryoid bodies. Cocultures of MS-5 stromal cells with ES cells were slightly more productive than HGFs. A strong synergistic effect was observed on the growth of myeloid colonies and MKs when we used a combination of MS-5 cells plus the HGF cocktail. Conditioned medium from MS-5 cells also synergized with the HGF cocktail to produce a substantial number of mixed colonies containing MKs. The addition of fibroblast growth factor-2 (FGF-2) to the HGF cocktail plus MS-5 nearly doubled the number of myeloid progenitors, including those with MKs. Thrombopoietin (TPO) alone or in any combination with MS-5 or HGFs, did not increase the number of MK-containing colonies. However, when TPO was added to the HGF cocktail + FGF-2 + MS-5, the number of MKs in liquid cultures and mixed colonies increased, and many exhibited a "hairy" appearance resembling pseudopodial proplatelet formation. Having defined the culture conditions of ES cells that allow the production of all the myeloid lineages including MKs, we conclude that the hematopoietic differentiation model of ES cells is especially useful for studying the regulation of expression of any gene important in early hematopoiesis.

Animals↗

PubMed1996

Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps.

The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent embryonic stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study, we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial differentiation occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM), Flk-1, tie-1, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation. PECAM and tie-2 mRNAs were found to be expressed only from day 4, whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-1, PECAM, VE-cadherin, MECA-32, and MEC-14.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell differentiation. The addition of an angiogenic growth factor mixture including erythropoietin, interleukin-6, fibroblast growth factor 2, and vascular endothelial growth factor in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis.

Animals↗

PubMed1996

The tissue-specific transcriptional regulation of the megakaryocytic glycoprotein IIb gene is controlled by interactions between a repressor and positive cis-acting elements.

Much information on regulation of the transcription of megakaryocytic genes stems from studies on the glycoprotein IIb (GPIIb) gene, an early and specific marker of this lineage. Transcriptional activity is controlled by the association of positive promoter elements corresponding to binding sites for the transcription factor GATA-1 and a member of the Ets family. In the present study, we show that these elements are not directly involved in the control of cell specificity. In contrast, we identified a sequence located between -170 and -73 that exhibited a repressor activity based on an analysis of the transcriptional activity of 5'-deleted GPIIb promoter fragments transfected in the nonhematopoietic HeLa cells. Further analysis of this repressor by substitution mutagenesis of the -139/-63 region showed that bases -120/-116 and -102/-93 were required for full repressor activity. The repressor is able to interact differentially with GPIIb promoter elements active in the megakaryocytic HEL, the erythroid K562, the monocytic U937, or the nonhematopoietic HeLa cell lines, indicating that it controls GPIIb gene tissue specificity. In addition, direct evidence for tissue-specific interaction between this repressor and the GPIIb -598/ -406 enhancer was obtained when these elements were set in the context of a heterologous SV40 promoter. Interestingly, the same repressor element controlling tissue specificity of the GPIIb gene may also control its temporal expression during megakaryocyte differentiation, based on recent evidence obtained by Fong and Santoro (J Biol Chem 269:18441, 1994). Finally, we found that the -120/-116 GPIIb sequence was part of a consensus motif shared by promoters of other megakaryocyte-specific genes, suggesting a common repressor mechanism.

Base Sequence↗

PubMed1996

Transcriptional regulation of the human CD9 gene: characterization of the 5'-flanking region.

The CD9 antigen, initially discovered on B lineage leukemic cells, belongs to the tetraspan superfamily of surface molecules. If no precise function has been assigned to any of these molecules, there are some indications that they could be involved in cell adhesion and cell migration, as well as malignant progression. The CD9 antigen is associated with surface proteins such as VLA integrins or HB-EGF precursor. Transfection of CD9 in melanoma cells reduces tumor growth and metastasis. The heterogenous distribution of the CD9 antigen suggests a complex regulation of its expression. We have previously characterized the CD9 gene and shown that transcription could be initiated at several sites in the TATA-less 5'-flanking region. We show here, using as a model two human leukemic cell lines with erythromegakaryocytic potential, HEL and K562, that the [-205, -154] region supports a promoter activity when cloned ahead of a CAT reporter gene. Mutagenesis analysis suggested the presence of a positive element located within the [-170, -154] region. Gel shift experiments using HEL extracts were compatible with the binding of the transcriptional factor Sp1 to the [-237, -205] region and indicated that a non-identified protein binds to the 3' end of the [-205, -154] region.

Antigens, CD↗

PubMed1996

Hematopoietic differentiation of embryonic stem cells: an in vitro model to study gene regulation during megakaryocytopoiesis.

We are interested in the regulation of the tissue specificity of the megakaryocyte-specific platelet glycoprotein IIb gene. The murine embryonic stem (ES) cells are able to differentiate into erythroid, mast and granulomonocytic cells in appropriate culture conditions. Our goal is to optimize the production of myeloid cells including megakaryocytes (MKs) by ES cells. We have found that coculture with MS-5 stromal cells and the presence of a cocktail of hematopoietic growth factors (HGFs) [stem cell factor, interleukin 3 (IL-3), IL-6, IL-11, G-CSF and erythropoietin] had a high synergistic activity on differentiation of ES cells into pure and MK-containing myeloid colonies from day 12 embryoid bodies. Thrombopoietin increased the number of MKs only when added to the HGF cocktail in the presence of MS-5 cells. Interestingly, many MKs exhibited a "hairy" appearance evocative of pseudopodial proplatelet formation. Expression of genes specific for the megakaryocytic lineage, GPIIb, PF4, mpl and GPIIIa, was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) during differentiation of ES cells, and their relative time course was evaluated. This demonstrates that optimized culture conditions for the differentiation of ES cells into the MK lineage provide a useful tool for the study of the regulation of expression of genes during megakaryocytopoiesis.

Animals↗

PubMed1996

A nonsense mutation in the GPIIb heavy chain (Ser 870-->stop) impairs platelet GPIIb-IIIa expression.

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.

Adolescent↗

PubMed1995

Regulation of gene transcription during the differentiation of megakaryocytes.

Glycoprotein IIb (GPIIb) is an early and specific marker of the megakaryocytic lineage. Thus studies on the transcriptional regulation of this gene may provide helpful information on the mechanisms controlling cell specificity and differentiation of this lineage. In previous experiments, the promoter of GPIIb gene was isolated and we have shown that a fragment extending 643 bp upstream the transcription start site was able to control the cell specificity of a reporter gene in transfection experiments of different permanent cell lines. Most of the transcriptional activity is contained in an enhancer containing binding sites for members of the GATA and ets transcription factors families. The transcription factor GATA1 is not only a major regulator of the transcription of erythroid genes, but it also regulates the expression of GPIIb and other megakaryocytic genes. We suggest that the lineage specificity and the temporal activation of GPIIb gene during hematopoiesis rely on the activity of a repressor that has been identified on the promoter. To test this hypothesis, we have developed a cell model allowing the study of the megakaryocytes differentiation from very immature progenitors to fully differentiated cells. This model is based on the differentiation of mouse embryonic stem cells. We have obtained megakaryocytes together with erythrocytic and granulo-macrophagic cells. The transfection in these ES cells of GPIIb promoter constructs mutated or not on different regions, including the repressor element will provide important information on the mechanisms controlling gene activation or repression during megakaryocyte differentiation.

Animals↗

PubMed1993

The transcription factor GATA-1 regulates the promoter activity of the platelet glycoprotein IIb gene.

Glycoprotein IIb (GPIIb) is an early and specific marker of the megakaryocytic lineage. We have previously shown that a fragment extending 643 base pairs upstream the transcription start site of the human GPIIb promoter was able to control the tissue-specific expression of the CAT gene in transfection experiments. Four potential GATA-binding sites, located at positions -463, -376, -243, and -54 are present within this fragment. Gel shift analysis revealed that nuclear extracts from the erythroleukemic cell line HEL contain a DNA-binding protein that recognizes these GATA sites. Using an antiserum raised to an hydrophilic region of the transcription factor GATA-1, the HEL GATA-binding protein was found to be GATA-1. Point mutations of the different GATA sites indicated that they did not equally contribute to GPIIb promoter activity. The -463 GATA motif located in an enhancer region is essential for full transcription activity and was found to be dominant upon the other GATA motifs. When this site is mutated, the -54 GATA site appears to be essential for the remaining CAT activity. These results indicate that the transcription factor GATA-1 plays an important role in the regulation of the transcription of the megakaryocyte specific GPIIb gene.

Amino Acid Sequence↗

PubMed1993

Organization of the human CD9 gene.

The CD9 antigen was originally described as a 24-kDa molecule present on B-lineage-derived acute lymphoblastic leukemia cells and developing B lymphocytes. Platelets also express a large amount of CD9 antigen and can be activated by CD9 antibodies. We report here the structure of the CD9 gene, which is composed of 8 exons spanning more than 20 kb. There is no TATA or CAAT box in the 5'-flanking domain of the CD9 gene, but a 120-bp region extremely rich in C and G (88%) contains several Sp 1 binding sites and a consensus site for the binding of zinc-finger proteins of the Krox/EGR family. The CD9 antigen belongs to a new cell surface protein family. The organization of its gene closely resembles the organization of the genes for two other members of this protein family, TAPA1 and CD63, which share with CD9 respectively 45 and 25% identity at the amino acid level.

Amino Acid Sequence↗

PubMed1992

Characterization of a specific erythromegakaryocytic enhancer within the glycoprotein IIb promoter.

The gene coding for glycoprotein IIb (GPIIb), the alpha subunit of platelet integrin GPIIb/IIIa is an early and specific marker of the megakaryocytic lineage. Thus, studies on the regulation of this gene may provide helpful information on the mechanisms controlling cell specificity and differentiation in this lineage. The promoter region of this gene was isolated and analyzed to understand its tissue-specific transcriptional activity. A region extending from nucleotides -414 to -554 was found to be extremely important for the promoter function. Deletion of this region results in a 70% decrease of the promoter activity, as measured in CAT assays. This region has the properties of an enhancer. It is able to activate a heterologous promoter, in a distance- and orientation-independent manner, in both megakaryocytic and erythroid cells. This enhancer contains binding sites for nuclear factors and mutation of these sites, individually or together, abolish the enhancer activity. These nuclear factors are present in megakaryocytic and erythroid cell lineages, but they are absent in the other tested cells. One of the sites, named domain D, contains a TTATC motif that may interact with the transcription factor GATA1, active in erythroid and megakaryocytic cells. These results indicate that the promoter of a megakaryocytic gene contains a tissue specific enhancer, active in both the erythroid and the megakaryocytic lineages, and may implicate the erythroid factor GATA1.

Base Sequence↗

PubMed1991

Tissue-specific expression of the platelet GPIIb gene.

One of the major objectives in the study of thrombogenesis is to determine the mechanisms by which a hematopoietic progenitor is activated and committed to the megakaryocytic lineage. Recent development of primary cultures of human megakaryocytes and the molecular cloning of genes that are specific to this lineage offer the possibility of getting some insights into the genetic mechanisms that control megakaryocytopoiesis. One gene of interest is the glycoprotein IIb (GPIIb) gene; GPIIb, the alpha subunit of the platelet cytoadhesin GPIIb-IIIa, is produced in megakaryocytes at an early stage of the differentiation, whereas the other subunit of this complex, GPIIIa, is expressed in other cells. For these reasons, the 5'-flanking region of the GPIIb gene was used to identify the regions that interact with DNA-binding nuclear factors. A fragment extending from -643 to +33 is capable of controlling the tissue-specific expression of the CAT gene in transfection experiments. Within this region, we have identified several sequences that are implicated in DNA protein interactions as shown in DNAse I footprints and gel mobility shift assays. One region, centered at -54, is similar to a nuclear factor E1-binding site, and a region located at position -233 contains a CCAAT motif. Two domains centered at positions -345 and -540, respectively, bind proteins that are present in megakaryocytic cells and nonrelated cells as well. Finally, two other domains, located at positions -460 and -510, interact with proteins that are only present in megakaryocytic cells. In addition, deletion of the region containing these two domains results in a significant decrease of the promoter activity. It is very likely that these domains bind megakaryocyte-specific nuclear proteins acting as positive transcription factors.

Base Sequence↗