An Sp1-binding Silencer Element Is a Critical Negative Regulator of the Megakaryocyte-specific αIIb Gene

Abstract

The Sp1 family of transcription factors are often involved in the regulated expression of TATA-less genes, frequently enhancing gene transcription. In this paper, we demonstrate that an Sp1-binding element inhibits the expression of the megakaryocyte-specific ␣ IIb gene in all cell lines tested and that this inhibition is actively overcome only in megakaryocyte-like cell lines. We had noted previously in primary megakaryocytes that a 50-base pair (bp) deletion from ؊150 to ؊101 bp in the rat ␣ IIb promoter region resulted in increased expression. We now show that deletion of this region markedly increased expression in both megakaryocytic and nonmegakaryocytic cell lines, eliminating the tissue specificity of the ␣ IIb promoter. Electrophoretic mobility shift assays (EMSA) defined a single complex, which bound to a ؊145 to ؊125 bp subregion. Point mutations within this region, localized the critical point of binding around bases ؊136/؊135, and expression studies showed that introduction of the ؊136/؊135 mutation into the rat ␣ IIb promoter had a comparable result to that seen with the 50-bp deletion. EMSA studies with the homologous human ␣ IIb promoter region gave an identical migrating band. Southwestern blots of HeLa nuclear proteins with both the rat ؊145 to ؊125 DNA and its human homologue bound to a single ϳ110-kDa protein, the known molecular weight of Sp1. Confirmation that this region of the ␣ IIb gene promoter bound Sp1 was accomplished using EMSA studies with an Sp1 consensus probe, antiSp1 and -Sp3 antibodies, and recombinant Sp1 protein.Further support for the role of Sp1 in the silencing of the ␣ IIb promoter was obtained using a Gal4 binding site substitution for the silencer region of ␣ IIb and co-expression of near full-length Sp1/Gal4 fusion protein expression vectors. Ectopic reinsertion of the ؊150 to ؊101 bp region, back into the ؊150 to ؊101 bp deleted promoter, enhanced rather than decreased expression, suggesting that Sp1's inhibitory role at ؊136/؊135 depends on its local interactions. In summary, we believe that we have identified a cross-species, non-consensus Sp1-binding site that binds Sp1 and that acts as a silencer of ␣ IIb expression in many cell lines. A model is presented as to how this Sp1-binding silencer element contributes to the megakaryocyte-specific expression of ␣ IIb gene.Platelets have a central role in thrombus formation. These anuclear cytoplasmic fragments are derived from bone marrow megakaryocytes and are highly differentiated (1). One of the specialized features found on the platelet membrane is the ␣ IIb /␤ 3 (also known as glycoprotein IIb/IIIa or CD41b) integrin receptor (2). This receptor is densely packed on the platelet surface. Following platelet activation, this receptor binds fibrinogen and plays an important role in platelet aggregation (2). Normally, ␣ IIb /␤ 3 is only found on developing megakaryocytes and circulating platelets. This is due to the tissue-specific nature of the ␣ IIb subunit. While ␤ 3 is expressed in several different cell types (3), ...

New Functional Promoter Polymorphism, CETP/−629, in Cholesteryl Ester Transfer Protein (CETP) Gene Related to CETP Mass and High Density Lipoprotein Cholesterol Levels

“…30,50 Indeed, a recent study involving in vitro transient transfection has revealed an interaction between 2 transcription factors, Sp1 and GATA 1. 48 Together, these data suggest that despite its distal position, the Ϫ629-Sp1 binding site may modulate transcriptional activity of the CETP gene by synergistic interaction with other nuclear factors, including ARP-1, SREBP-1, and C/EBP.…”

“…There is some evidence that Sp1 may itself decrease transcriptional activity when bound to certain Sp1 elements, depending on its local interactions. 48 On the other hand, it has been demonstrated that Sp3 is a bifunctional protein containing independent repressor and activator domains. Such dualfunction regulation is dependent on the promoter and the cellular context.…”

A novel T-77C polymorphism in DNA repair gene XRCC1 contributes to diminished promoter activity and increased risk of non-small cell lung cancer

“…Confirmed by the EMSA and ChIP assays, the transcriptional factor Sp1 indeed preferentially binds to the À77C allele but, if any, very weakly to the À77T allele in vitro and in vivo. Sp1 is generally considered as a transcriptional activator; however, as demonstrated in many previous studies, Sp1 may also function as a transcriptional inhibitor, repressing gene transcription in certain promoter contexts (Pagliuca et al, 1998;Shou et al, 1998;Dean et al, 2000;Li and Ou, 2001;Won et al, 2002). As XRCC1 plays important roles in BER (Thompson et al, 1990;Tebbs et al, 1999;Weinfeld et al, 2001;Caldecott, 2003), reduced expression of the protein would be expected to impair BER ability and hence increase the risk of lung cancer.…”

Transcriptional Activity of Sp1 Is Regulated by Molecular Interactions between the Zinc Finger DNA Binding Domain and the Inhibitory Domain with Corepressors, and This Interaction Is Modulated by MEK

“…Indeed, many reports have implied that Sp1 and the Sp1-like transcription factors exhibit a bipolar character; i.e. they function as either transcription activators or repressors, depending on the promoter to which they bind and the coregulators with which they interact (1, 2, 9, 12-14, [23][24][25][26][27].…”