Functional analysis of SOX3 binding at the Dbx1 locus.

Abstract

Sox3 a members of the SOX transcription factor family, is essential for normal brain development and required for growth of pituitary and hypothalamus. Sox3, as well as Sox2 which is another member in SOXB1 subfamily are widely expressed in neural progenitor cells and show functional redundancy. ChIP-seq data by Bergsland et al, 2011 has identified five putative SOX3 binding sites near/at the Dbx1 locus. Microarray data from the lab (N. Rogers, unpublished data) has identified Dbx1 as downregulated in Sox3 null neural progenitor cells. Together these data suggest that Dbx1 may be regulated directly by SOX3. To investigate the possibility that SOX3 regulates the Dbx1 locus in vitro, we performed gel shift assays and Luciferase Reporter Assays to see if SOX3 binds any of the five Dbx1 regulatory sites. Due to time constraints we were not able to optimize the gel shift assays to obtain any informative results. Secondly, we optimized Luciferase Reporter Assays providing preliminary data suggesting SOX3 may bind at one of the tested Dbx1 sites. To study the redundancy between Sox2 and Sox3, Sox2 was also tested in the Luciferase Reporter Assays indicating Sox2 may also regulate the same site as Sox3 . Due to time constraints, the other three binding sites remain to be analyzed in the future. The function of Dbx1 is best characterized in the context of the developing neural tube (also known as the spinal cord). To identify how other neural tube marker genes are regulated by Sox3, qPCR was performed with some marker genes in Sox3 null E9.5 mouse embryos compared with WT embryos. Dbx1, Pax6, Ngn2 and Olig2 all showed significant decrease in Sox3 null. Further study of these genes will be required to assess the significant outcome of their down regulation in an in vivo context.