TG101348 In this study we present a novel regulatory interac

In this study we present a novel regulatory interaction between SOX2 and the TRKC gene. We show that SOX2 is important for TRKC expression as the loss of SOX2 effectively decreases TRKC expression. We also find that SOX2 regulates expression by directly interacting with SOX-specific binding motifs within the TRKC regulatory region. We demonstrate that these sites are conserved among several mammalian species and are important for TRKC gene transcription. Our study is the first to identify a role for SOX2 in the regulation of the TRKC gene in hES TG101348 and suggests a pathway by which SOX2 regulates the maintenance of the stem cell state in these cells.
Results
Discussion/conclusions The expression of the TRK receptors in hES cells proved to be an important finding in improving single cell culture conditions. The dissociation of hES cells into single cells for genetic manipulations renders them particularly susceptible to apoptosis. Previous studies have found that the disruption of E-cadherin signaling responsible for cell–cell interactions may be the cause for single cell death (Xu et al., 2010). The addition of the ligands of TRKB and TRKC, however, is able to improve clonal cell survival in suboptimal growth conditions, such as during genetic manipulations (Hohenstein et al., 2008). Although the genes encoding the TRK receptors are well studied in neuronal cells and play important roles in cell fate determination, growth and proliferation, they are not well-studied in hES cells. Due to the receptors\' role in improving hES cell survival, understanding how the genes for the receptors are regulated would enhance our knowledge of hES cell biology. The transcription factor SOX2 was shown to be essential in maintaining pluripotency and self-renewal in hES cells. Downregulation of this gene results in the loss of expression of many genes important in preserving the stem cell state (Fong et al., 2008). Since TRKC has been shown to be important for maintaining hES cell survival, we sought to determine if SOX2 could regulate TRKC expression in hES cells. Our results show that hES cells do indeed express the genes for the TRKB and TRKC receptors. Both the full-length and truncated splice variants of the TRKC gene were expressed at much higher levels than any splice variant of TRKB. Only the truncated variant of TRKB was expressed at a very low level. Previous studies have shown that truncated forms of the TRKC receptor can still induce ligand-dependant signaling in addition to the canonical signaling pathway (Esteban et al., 2006). The high expression of both forms of TRKC indicates that the gene may be important in maintaining survival and self-renewal in hES cells. Our results also show that the expression level of TRKC is regulated by SOX2, as the loss of SOX2 resulted in considerable downregulation of the TRKC gene. We also observed a decrease in TRKC by immunocytochemistry indicating that the loss of SOX2 was indeed reducing the amount of TRKC protein present on the cell surface. The expression levels of TRKB, however, were not significantly affected suggesting that SOX2 may not be involved in the regulation of TRKB or that the level of SOX2 reduction was insufficient in downregulating TRKB. Analysis of the regulatory region of TRKC identified two evolutionarily conserved SOX binding motifs. Our results from the ChIP assays demonstrate that SOX2 interacts at this specific region on the genome termed SOX Site A which contains a SOX binding site. It also interacts minimally at a second site termed SOX Site B. However, the level of enrichment at the Site B region is approximately 3-fold less than at Site A indicating that Site A likely plays a greater role in the regulation of TRKC in hES cells. The specificity for which SOX2 interacts with the TRKC gene and the conservation of the SOX2 site indicates that this site is important for regulation of the TRKC gene. We also found that Site A on the TRKC promoter is important for transcription of the TRKC gene as shown by luciferase assays. Transfection of hES cells with TRKC-LUC Site A reporter vector increased the fold change in luciferase activity by approximately 6-fold compared to cells transfected with just the EMPTY-LUC vector. Including Site B in the reporter construct, however, only increased luciferase activity slightly over the EMPTY-LUC control. This difference in luciferase activity with the inclusion of Site B could be due to the ability of the site to bind repressors. In addition, it is likely that some differences may exist between the endogenous TRKC promoter and the TRKC promoter elements contained within the reporter construct. Thus the construct may not behave in a manner which entirely reflects the activity of the endogenous gene in hES cells. Deletion of Site A, however, in hES cells transfected with the TRKC-LUC Site delA construct decreased activity to approximately 2-fold indicating that Site A is needed to drive expression of the reporter gene. In the HFF-1 cells, expression of SOX2 was able to drive expression of the reporter gene in the TRKC-LUC Site A vector. Deletion of Site A, however, dramatically decreased luciferase activity indicating that SOX2 utilizes Site A to activate the TRKC promoter. Interestingly, we observed an increase in luciferase activity in HFF-1 cells transfected with the TRKC-LUC Site A+B construct in contrast to the activity observed when the construct was transfected in hES cells. The disparity in activity may be due to repressors binding to Site B present in hES cells that are not present in the HFF-1 cells. However, further studies are required to determine the activity at this site. Overall, our results in conjunction with the results of the ChIP assay show that SOX2 utilizes Site A to produce a positive transcriptional effect on the TRKC gene.