An Sp1-binding silencer element is a critical negative regulator of the megakaryocyte-specific alphaIIb gene

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 alphaIIb 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 alphaIIb promoter region resulted in increased expression. We now show that deletion of this region markedly increased expression in both megakaryocytic and non-megakaryocytic cell lines, eliminating the tissue specificity of the alphaIIb 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 alphaIIb promoter had a comparable result to that seen with the 50-bp deletion. EMSA studies with the homologous human alphaIIb 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 approximately 110-kDa protein, the known molecular weight of Sp1. Confirmation that this region of the alphaIIb gene promoter bound Sp1 was accomplished using EMSA studies with an Sp1 consensus probe, anti-Sp1 and -Sp3 antibodies, and recombinant Sp1 protein. Further support for the role of Sp1 in the silencing of the alphaIIb promoter was obtained using a Gal4 binding site substitution for the silencer region of alphaIIb 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 alphaIIb expression in many cell lines. A model is presented as to how this Sp1-binding silencer element contributes to the megakaryocyte-specific expression of alphaIIb gene.