An Ets/Sp1 interaction in the 5'-flanking region of the megakaryocyte-specific alpha IIb gene appears to stabilize Sp1 binding and is essential for expression of this TATA-less gene.

The megakaryocyte-specific integrin, alpha IIb, is the alpha-subunit of the alpha IIb/beta 3 complex found on the surface of platelets. This complex is a receptor for fibrinogen and other ligands when platelets are activated. Because the alpha IIb gene is specifically expressed in megakaryocytes, the 5'-flanking region was studied as a potential model for megakaryocyte-specific gene expression. Previous studies have defined some of the important regulatory elements in the 5'-flanking region of this gene. The present studies focus on the issue of the molecular basis by which this TATA-less gene is properly transcribed. A GA-rich region centered 14 bp upstream from the transcriptional start site appears to be a nonconsensus Sp1-binding site. Binding to this site is of low affinity, but is markedly improved by interaction with protein(s) binding at an Ets-consensus site approximately 20 bp further upstream. Mutation of the Ets site greatly reduces the ability of Sp1 to bind to its site. Trans-acting nuclear factors binding to and interaction of the proteins at these two sites have direct effects on the observed promoter activity in primary megakaryocyte transient expression studies. These studies provide further evidence of the role of interactions between Ets-like proteins and Sp1 in transcriptional activation when a TATA box is not present in the promoter region of a gene. Based on the presented studies and previous results, a model is proposed for the regulation of expression of the alpha IIb gene.

[1]  O. Bernard,et al.  GATA-and SP1-binding sites are required for the full activity of the tissue-specific promoter of the tal-1 gene. , 1994, Oncogene.

[2]  M. Rechler,et al.  Three clustered Sp1 sites are required for efficient transcription of the TATA-less promoter of the gene for insulin-like growth factor-binding protein-2 from the rat. , 1993, The Journal of biological chemistry.

[3]  R. Tjian,et al.  Transcription from a TATA-less promoter requires a multisubunit TFIID complex. , 1991, Genes & development.

[4]  J. George,et al.  Glanzmann's thrombasthenia: the spectrum of clinical disease. , 1990, Blood.

[5]  D. Seshasayee,et al.  Functional interaction of GATA1 with erythroid Krüppel-like factor and Sp1 at defined erythroid promoters. , 1996, Blood.

[6]  R. J. Fisher,et al.  An intricate arrangement of binding sites for the Ets family of transcription factors regulates activity of the alpha 4 integrin gene promoter. , 1994, The Journal of biological chemistry.

[7]  G. Uzan,et al.  The transcription factor GATA-1 regulates the promoter activity of the platelet glycoprotein IIb gene. , 1993, The Journal of biological chemistry.

[8]  P. Frachet,et al.  Isolation of the human platelet glycoprotein IIb gene and characterization of the 5' flanking region. , 1988, Biochemical and biophysical research communications.

[9]  T. Inoue,et al.  Biological and biochemical characteristics of murine megakaryoblastic cell line L8057. , 1993, Experimental hematology.

[10]  P. Romeo,et al.  GATA and Ets cis-acting sequences mediate megakaryocyte-specific expression , 1993, Molecular and cellular biology.

[11]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[12]  K. A. Lee,et al.  A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. , 1988, Gene analysis techniques.

[13]  G. Uzan,et al.  Tissue-specific expression of the platelet GPIIb gene. , 1991, The Journal of biological chemistry.

[14]  Characterization of a new megakaryocytic cell line: the Dami cell. , 1988 .

[15]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[16]  M. Poncz,et al.  Platelet glycoprotein IIb gene expression as a model of megakaryocyte‐specific expression , 1995, Stem cells.

[17]  Gregory Riely,et al.  Isolation and characterization of a TATA-less promoter for the human beta 3 integrin gene. , 1994, Blood.

[18]  W. Min,et al.  Functional Analysis of the Human Endothelial Nitric Oxide Synthase Promoter , 1995, The Journal of Biological Chemistry.

[19]  B. Hirt.  Selective extraction of polyoma DNA from infected mouse cell cultures. , 1967, Journal of molecular biology.

[20]  M. Klemsz,et al.  Hematopoietic lineage- and stage-restricted expression of the ETS oncogene family member PU.1. , 1993, Blood.

[21]  K. Ravid,et al.  Characterization of regulatory elements in the 5'-flanking region of the rat GPIIb gene by studies in a primary rat marrow culture system. , 1994, Blood.

[22]  M. Klemsz,et al.  The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene , 1990, Cell.

[23]  O. Farokhzad,et al.  The human beta 2 integrin CD18 promoter consists of two inverted Ets cis elements , 1994, Molecular and cellular biology.

[24]  M. Lieberman,et al.  In vitro establishment and characterization of a human megakaryoblastic cell line , 1990 .

[25]  D. Tenen,et al.  The Sp1 transcription factor binds the CD11b promoter specifically in myeloid cells in vivo and is essential for myeloid-specific promoter activity. , 1993, The Journal of biological chemistry.

[26]  R. Eisman,et al.  Organization of the gene for platelet glycoprotein IIb. , 1990, Biochemistry.

[27]  E. Prochownik,et al.  Regulation of megakaryocyte phenotype in human erythroleukemia cells. , 1990, The Journal of clinical investigation.

[28]  A. Haese,et al.  Cooperation of GATA-1 and Sp1 can result in synergistic transcriptional activation or interference. , 1993, The Journal of biological chemistry.