Transcriptional regulation of tissue-specific expression of the platelet [alpha]IIb gene

The integrin [alpha]IIb gene is expressed in a megakaryocyte-specific fashion. This dissertation study has characterized an Sp1-binding silencer element between -145 bp and -125 bp in the immediate promoter region of the rat [alpha]IIb gene that may contribute to the tissue-specific nature of the [alpha]IIb gene expression. Transient transfection studies showed that deletion of this element not only resulted in increased reporter gene expression in megakaryocytic cell lines but also induced high levels of expression in non-megakaryocytic cell lines that normally do not express [alpha]IIb gene. By electrophoretic mobility gel shift assays (EMSA), it was shown that a single nuclear complex was formed ubiquitously at this site in all the cells tested. A Southwestern blot and EMSA studies using an Sp1-consensus probe, anti-Sp1 and anti-Sp3 antibodies, and recombinant Sp1 protein consistently suggested that this silencer element bound Sp1. Moreover, by point mutation studies, the Sp1-binding activity at this site was shown to correlate directly with its silencing effect on expression. An identical nuclear complex involving Sp1 formed with the homologous human [alpha]IIb promoter region, suggesting the conservation of this silencer element between species. It was also found that the silencing activity of this element was position-dependent. It suggests that local interactions between Sp1 and other bound nuclear proteins may be important for the negative regulation of the [alpha]IIb gene expression by this silencer element. It is proposed that Sp1 bound to the silencer site silences the [alpha]IIb gene in all tissues, and only in megakaryocytic cells this inhibition is overcome by additional sequences further upstream that may bind tissue-specific factors. This dissertation also presents transient expression data from both cell lines and primary rat bone marrow culture that a 3 kb 5'-flanking region of the rat [alpha]IIb gene appears to have similar tissue-specificity and promoter strength with the 912 bp of the [alpha]IIb promoter sequence. The establishment of five transgenic mouse lines for a prokaryotic [beta]-gal reporter gene driven by the 912 bp of the rat [alpha]IIb promoter is also reported in this dissertation. The megakaryocyte-specific expression of the transgenic [beta]-gal gene was not seen in all these lines tested by X-Gal staining. It suggests that the 912 bp promoter region may not be adequate to direct high-levels of tissue-specific expression of the [alpha]IIb gene in a chromosomal environment.