A dominant negative over expression model of mammalian MED12 function

Although schizophrenia has been shown to have a substantial component, there is a dearth of known risk alleles. Furthermore, all of the known risk genes are of small effect sizes. Previously it has been shown that a 12 base pair insertional polymorphism in the in the C-terminal, opposite paired (Opa) domain of the MED12 gene known as MED12 represents a small but significant risk for a positive syndrome psychosis. In addition, the MED12 polymorphism is found in approximately 1.6 percent of Xchromosomes of northern European decent. Studies in zebrafish show that alterations of MED12 reduce staining for monoaminergic neuronal populations, including dopaminergic and serotonergic populations. However, precise mechanisms through which these changes occur remain unknown. For this study PC6-3 cells were used as a mammalian, cell culture model for studying cellular and transcriptional effects of MED12 in a dopaminergic cell type. This approach was based on studies that have shown that overexpression of C-terminal MED12 proline, glutamine and leucine rich (PQL) and Opa domain constructs interact in a dominant negative manner with several transcriptional regulatory proteins that interact with MED12. GFP tagged PQL and Opa domain constructs were placed into a tetracycline inducible T-RExTM regulated expression vector and introduced into a previously generated PC6-3, TR156 cell line expressing the Tet-Repressor molecule. In this study, I report a selection bias against stably transfected cell lines strongly expressing constructs containing the two C-terminal PQL-Opa protein domains of MED12. I also show that the described low levels of induction of that construct are associated with small, but significant alterations in nuclear morphology, possibly due to nuclear reorganization. Induction of PQL-Opa domains increases in cell metabolism by an MTS, tetrazolium salt assay typically associated with increased proliferation compared to the Opa domain alone. Interestingly, the MTS results in the stable cell lines were not