Transcription Factor Interactions Governing Gene Regulation by the Wnt Signalling Pathway

Transcriptional regulation by Wnt signalling is primarily thought to be accomplished by a complex of β-catenin and TCF family transcription factors (TFs). Although numerous studies have suggested that additional TFs play roles in regulating Wnt target genes, their mechanisms of action have not been investigated in detail. This dissertation primarily examines the role of two developmentally important sets of transcription factors – CDX and SOX proteins, in particular the family member SOX9, their interactions with Wnt/β-catenin signalling, and their importance in gene regulation. I characterised a Wnt-responsive element (WRE) downstream of the Wnt target gene Axin2 and found that TCFs and Caudal type homeobox (CDX) proteins were required for its activation. Using a new separation-of-function TCF mutant, I found that WRE activity requires the formation of a TCF/CDX complex. Systematic mutagenesis of this enhancer identified other sequences essential for activation by Wnt signalling, including several copies of a novel CAG DNA motif. Computational and experimental evidence indicates that the TCF/CDX/CAG mode of regulation is prevalent in multiple WREs. Together, these results demonstrate the complex nature of cis- and trans- interactions required for signal-dependent enhancer activity. The transcription factor SOX9 potently represses many Wnt target genes by down-regulating β-catenin protein levels. I found that SOX9 surprisingly promotes the proliferation of Wnt-driven colorectal cancer (CRC) cells. In contrast to how it indirectly represses Wnt targets, SOX9 directly co-occupies and activates multiple Wnt-responsive enhancers in CRC cells. Examination of the binding site grammar of these enhancers shows the presence of TCF and SOX9 binding sites that are necessary for transcriptional activation. In addition, I identified a physical interaction between the DNA-binding domains of TCFs and SOX9 and showed that TCF-SOX9 interactions are important for target gene regulation and CRC cell growth. This work demonstrates a highly context-dependent effect of SOX9 on Wnt targets, with the presence or absence of SOX9 binding sites on Wnt-regulated enhancers determining whether they are directly activated or indirectly repressed by SOX9.PhDMolecular, Cellular, and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/175584/1/raravind_1.pd