Quantitative analysis of interactions between TCF and context transcription factor binding in Wnt target regulation

TCF is the Wnt pathway effector transcription factor, which works with other “context” transcription factors to regulate Wnt target genes. The goal of this project is to analyze endogenous cis-regulatory elements that are regulated by Wnt signaling during C. elegans development, and compliment our findings with studies of synthetic regulatory elements designed and synthesized in the lab. Amanda previously identified ten cis-regulatory elements for 6 different genes that are the targets of Wnt signaling. We are currently working to identify the other transcription factors that act as context inputs for these enhancers. We will evaluate the organization of the different enhancers with regard to binding site number, strength, orientation and spacing of context factor sites and TCF sites, along with predicted nucleosome positioning. We will design synthetic enhancers to probe how each of these properties affects target expression and test the resulting models by measuring the expression driven by endogenous enhancers with targeted mutations.

Funded by R00 GM111825

Mechanisms and consequences of Wnt as a transmitotic morphogen

In cells that receive a Wnt signal, β-catenin enters the nucleus where it acts as a co-activator with TCF to activate target genes. The ability to track cells and their daughters throughout development with our model system previously allowed us to uncover a novel phenomenon: Wnt-signaled daughter cells of a cell that previously received a Wnt signal have almost 2-fold more nuclear β-catenin than the Wnt-signaled daughter of a cell that did not receive a Wnt signal (Zacharias et al, 2015). We found this effect becomes more pronounced in later development and affects target gene expression. We normally think of a morphogen as a signal that can activate distinct target genes based on the level or length of the input signal, but in this case Wnt is acting across rounds of mitosis, so we have termed this phenomenon a “transmitotic morphogen”. We plan to investigate how TCF and β-catenin concentrations directly control target gene expression by creating a dimmer switch modulate the nuclear levels of TCF or β-catenin, using a single cell heat shock approach and a quantitative image analysis method called CC-RICS. We are also examining how cis-regulatory elements encode the response to different concentrations of TCF or β-catenin. Finally, we are using a targeted RNAi screen to uncover the mechanism for the transmitotic enrichment of β-catenin.