The Regulated Expression of a TATA‐Less, Platelet‐Specific Gene, α_IIb

Abstract

The megakaryocyte (MK)-specific integrin, alphaIIb, is the alpha-subunit of the alphaIIb/beta3 complex found on the surface of platelets. This complex is a receptor for fibrinogen and other ligands when platelets are activated. Because the alphaIIb gene is specifically expressed in MKs, this gene was studied as a potential model for MK-specific gene expression. Previous studies have defined some of the important regulatory elements in 912 bp of the immediate 5'-flanking region of this gene. These studies defined several important elements including two GATA-binding elements and an Ets-binding element. Using a primary rat marrow expression system, we demonstrated that one of the GATA-binding elements, -454 bp upstream of the transcriptional start site (GATA454), is critical for expression of the alphaIIb gene. A potential negative regulatory element was found between -100 and -200 bp upstream of both the rat and human alphaIIb genes. The biological basis by which this negative regulatory region effects expression is not well understood. Recent studies have focused on the issue of the molecular basis by which this TATA-less gene is properly transcribed. We found that a GA-rich region approximately 14 bp upstream from the transcriptional start site appears to be a nonconsensus Sp1-binding site that interacts with an Ets-consensus site approximately 20 bp further upstream. 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 alphaIIb gene. In studies looking at more distal regulatory elements, we have found, using the primary rat marrow expression system, that 2.9 kb of 5'-flanking alphaIIb sequence has as high a level of expression as the 912 bp construct. Whether either of these lengths of 5'-flanking region can result in tissue-specific expression in transgenic models is presently being investigated. In addition, while a published report suggests that the two genes alphaIIb and beta3 are physically linked within a 250 kb region of genomic DNA, analysis of yeast artificial chromosome clones and genomic pulsed field gel electrophoresis analysis are consistent with these two genes not being tightly linked and being >1 mb apart, suggesting that these two genes do not form a single, tissue-specific locus.

Transcription of the Transforming Growth Factor β Activating Integrin β8 Subunit Is Regulated by SP3, AP-1, and the p38 Pathway

“…Sp1-4 form a subgroup of the SP family that contain homologous activation domains and C-terminal zinc finger regions, which mediate DNA binding specificity (54). We limited our investigation to Sp1 and Sp3 because they are expressed in tissues where ␤8 is known to be expressed and have been shown to regulate expression of a wide array of integrin genes (55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65). Their functions partially overlap as demonstrated by their knock-out phenotypes in mice (66 -68).…”

“…Promoter studies of other integrins, such as ␣2, ␣3, ␣5, ␣6, ␣11, ␣IIb, ␤4, ␤5, CD11␤-␦, and CD18, have shown requirements for Sp1/Sp3 and/or AP-1 transcription factors and their cognate sites for expression (55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(77)(78)(79). In particular, the promoter of ITGAV (integrin ␣v), the gene that encodes the heterodimerization partner of integrin ␤8, is also regulated by Sp1 and Sp3 (65).…”

Ets transcription factors bind and transactivate the core promoter of the von Willebrand factor gene

“…In these cases, as well as when a single Ets binding site is present, binding of cooperative transcription factor(s) is necessary for speci®c transactivation. c-Ets-1 and c-Ets-2 transactivate a large number of genes through interactions with a variety of factors (reviewed by Tymms and Kola, 1994), like Fos/Jun , Sp1 (Ge gonne et al, 1993;Block et al, 1996), GATA-1 (Lemarchandel et al, 1993). Essential Ets binding motifs cooperate with GATA elements for the tissue-speci®c expression of megakaryocytic genes such as glycoprotein (GP)IIb (Block et al, 1996;Lemarchandel et al, 1993;Martin et al, 1993), GPIba (Hashimoto and Ware, 1995), platelet factor four (PF4, Ravid et al, 1991), thrombopoietin receptor (MPL, Deveaux et al, 1996).…”

Regulation of Ets function by protein–protein interactions

“…Similarly, it is known that Rb, which interacts with Elf1, physically interacts with histone deacetylase HDAC1 (MagnaghiJaulin et al, 1998). Another example is Sp1, which interacts with many Ets factors (Block et al, 1996;Dittmer et al, 1997;Eichbaum et al, 1997;Krehan et al, 2000)), and has been shown to interact with HDAC1 (Doetzlhofer et al, 1999), repressing transcription. TEL is able to recruit co-repressors such as SMRT and mSin3A resulting in transcriptional repression Fenrick et al, 1999).…”