Transcriptional Twister: characterizing the plasticity of a bipartite TCF binding motif. Hilary Cara Archbold¹, Ken M. Cadigan^1,2. 1) Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI; 2) Department of Molecular, Cellular and Developmental Biology , University of Michigan, Ann Arbor, MI.

   In Drosophila, TCF/pangolin acts as a major transcriptional regulator of Wingless (Wg, a fly Wnt) signaling in multiple events that shape the developing organism, and maintain stem cell populations necessary for adult tissue homeostasis. The ability of TCF to bind the correct regulatory elements is critical for proper spatio-temporal expression of target genes. How TCF mediates such a broad range of outcomes is poorly understood. The HMG domain of TCF binds the sequence SSTTTGWW, and previous research in our lab identified a second sequence GCCGCCR (the Helper site) which is required for activation of multiple Wg targets. Our data support a model where TCF binds HMG and Helper site pairs, via two closely spaced domains, the HMG and the C-Clamp. Surprisingly, spacing and orientation of these two motifs varies both within and between Wingless Response Elements (WREs). My research is focused on understanding how motif architecture regulates TCF binding and/or activity, and using this information to identify new WREs and target genes. Using a luciferase reporter gene system in Drosophila Kc cell culture, we have shown that in both synthetic and known target gene WREs, the optimal spacing of the Helper site is orientation specific, is relative to the HMG domain imposed bend of DNA, and can be located either up or downstream of the HMG site. Our bioinformatic analysis has also shown an enrichment of optimal pairs in regions identified as bound by TCF in fly embryos (using a ChIPseq data set from Junion et al. Cell 148: 473). We are currently investigating the correlation between activation levels and binding affinity, and are using site-specific integration to characterize reporter gene expression level and patterns driven by optimal and suboptimal motifs in transgenic Drosophila. In addition, with our awareness of optimal motif configuration, we have identified several novel candidate WREs which will be tested for Wg dependent activity in Drosophila.