| 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 MED1212bp represents a small but significant risk for a positive syndrome psychosis. In addition, the MED1212bp polymorphism is found in approximately 1.6 percent of X-chromosomes 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-REx(TM) 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 reflected changes in cell numbers by light microscopy cell counts, or changes in cell cycle distribution fluorescence activated cell sorting (FACS).;I also show microarray gene expression data for the described MED12 tetracycline inducible lines, and the same MED12 constructs transiently electroporated into PC6-3 cells. For both stable and transient experiments, the arrays were characterized by small fold changes, which could not be validated by RT-PCR. The stable arrays did not produce any robust findings. However, gene ontology (GO) data, from the transiently electroporated cells shows that 9 of the top 31 GO categories are related to changes in proliferation and cytoskeletal reorganization. Despite this trend, the data from the GoMiner analysis was above the level of significance (alpha = 0.05), as indicated by the false discovery rates (FDR > 0.3). Analysis of directionality of expression demonstrated significant evidence of skewing in the pattern of differential expression of annotated genes where with a tendency for the most significantly differentially expressed annotated probes to be more highly expressed genes in the electroporated GFP control condition cells expressing the PQL/Opa construct. This is also consistent with a broad overlap of the expression data with ChIP-seq data suggesting that the dominant negative effect may be spread over many MED12 regulated genes, in which case the low expression levels are particularly problematic. While the data from these experiments do not present a clear mechanism for MED12 function, they are informative in developing models of MED12 alteration, and potential improvements are discussed. |
