AN SP1-BINDING SILENCER ELEMENT IS A CRITICAL NEGATIVE REGULATOR OF THE MEGAKARYOCYTE-SPECIFIC ALPHA(IIB) GENE

The Sp1 family of transcription factors are often involved in the regu lated expression of TATA-less genes, frequently enhancing gene transcr iption, In this paper, we demonstrate that an Sp1-binding element inhi bits the expression of the megakaryocyte-specific alpha(IIb) gene in a ll cell lines tested and that this inhibition is actively overcome onl y in megakaryocyte-like cell lines, We had noted previously in primary megakaryocytes that a 50-base pair (bp) deletion from -1.50 to -101 b p in the rat alpha(IIb) promoter region resulted in increased expressi on. We now show that deletion of this region markedly increased expres sion in both megakaryocytic and nonmegakaryocytic cell lines, eliminat ing the tissue specificity of the alpha(IIb) promoter, Electrophoretic mobility shift assays (EMSA) defined a single complex, which bound, t o a -145 to -125 bp subregion, Point mutations within this region, loc alized the critical point of binding around bases -136/-135, and expre ssion studies showed that introduction of the -136/-135 mutation into the rat alpha(IIb) promoter had a comparable result to that seen with the 50-bp deletion, EMSA studies with the homologous human alpha(IIb) promoter region gave are identical migrating band, Southwestern blots or HeLa nuclear proteins with both the rat -145 to -125 DNA and its hu man homologue bound to a single similar to 110-kDa protein, the known molecular weight of Sp1. Confirmation that this region of the alpha(II b) gene promoter bound Sp1 was accomplished using EMSA studies with ar e Sp1 consensus probe, anti-Sp1 and -Sp3 antibodies, and recombinant S p1 protein, Further support for the role of Sp1 in the silencing of th e alpha(IIb) promoter was obtained using a GaI4 binding site substitut ion for the silencer region of alpha(IIb) and co-expression of near fu ll-length Sp1/Ga14 fusion protein expression vectors, Ectopic reinsert ion of the -150 to -101 bp region, back into the -150 to -101 bp delet ed promoter, enhanced Father than decreased expression, suggesting tha t 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 Spl-binding site that binds Sp1 and that acts as a silencer of alpha(IIb) expression in many cell lines, A model is presented as t o how this Sp1-binding silencer dement contributes to the megakaryocyt e-specific expression of alpha(IIb) gene.