A small number of extracellular signaling pathways control a diverse array of processes during development and homeostasis by altering transcription in target cells. An important question in the regulation of genes by signaling pathways is how the same signal can activate different targets in different cells. The Wnt signaling pathway and its role in development are conserved among all metazoans and disruption of the pathway causes developmental defects and disease in humans, including cancer. I propose to uncover the fundamental rules that determine which genes are regulated by the Wnt pathway in different embryonic contexts by analyzing the enhancers of Wnt target genes and investigating the expression of synthetic enhancers. I will leverage synthetic enhancer strategies pioneered in yeast to systematically study enhancer function for the targets of the Wnt pathway over the course of developmental time in C. elegans embryos. To do this, I will use a powerful automated lineage tracing system that measures dynamic expression patterns quantitatively in a live developing embryo;because C. elegans has an invariant lineage, I can directly compare expression between reporters on a cellular level and identify cell fates without the need for separate markers. In preliminary work, I identified sixteen transcription factors that are targets of the Wnt pathway and found that contrary to previous thinking, some targets required the Wnt effector transcription factor, TCF, only to repress expression in unsignaled cells, while others required TCF only to activate expression in signaled cells. Furthermore, I found that the response to Wnt signaling is not uniform throughout the embryo;instead, cells with parents who received a Wnt signal exhibit a stronger response to a Wnt signal than cells with unsignaled parents. This indicates the cells possess transmitotic memory of Wnt signaling, a novel finding. In the mentored phase of this study, I will characterize enhancers that are direct targets of Wnt signaling and determine how TCF and context transcription factors contribute to the regulation of Wnt target genes. In the first aim, I will identify sites bound by TCF during embryogenesis and evaluate the ability of TCF binding sites to drive expression in synthetic enhancers. In the second aim, I will characterize the enhancers of Wnt target genes, identify the context factors that co-regulate them, and use synthetic enhancers to determine how context factors sites combine with TCF binding sites to regulate expression. The data I gather and techniques I learn in the mentored phase will help me be successful in undertaking the third aim in the independent phase. For this third aim, I will use synthetic enhancers to identify the properties that are essential for Wnt targets, including the numbers, strength, orientation, and spacing of TCF and context factor binding sites. I will then predict and test the effect of targeted mutations on endogenous target enhancers and generate synthetic enhancers with novel expression patterns to demonstrate understanding of the underlying rules. This research will significantly advance our ability to identify Wnt target genes and predict their expression by sequence alone. Due to evolutionary conservation, some of the rules that govern Wnt regulation in C. elegans will likely be universal principles that also govern regulation of this and other important pathways in vertebrates. During the initial phase of this award, I will receive mentoring from my primary mentor, Dr. John Murray, my co-mentor, Dr. Klaus Kaestner, and two additional faculty members with relevant expertise. This mentorship committee will assist me with learning new techniques, including genome engineering, ChIP-seq, and bioinformatic enhancer identification that will be essential in my future career as an independent investigator in developmental genetics. My training will also include coursework, grant writing, and a mentored faculty position search to allow me to transition to an independent career. The proposed training will take place at the Perelman School of Medicine at the University of Pennsylvania, which possesses outstanding facilities, an excellent collaborative environment with many opportunities for postdoctoral fellows to present their research, and an office of Biomedical Postdoctoral Programs that organizes training in career development and responsible conduct in research.
Developmental gene expression and signaling pathways are dysregulated in many disease states including cancer and knowledge about gene regulation can be manipulated to induce stem cells to adopt the desired fates. This research contributes to the basic understanding of how non-coding DNA controls gene expression by identifying the rules that govern the regulation of target genes by the Wnt signaling pathway.
|Zacharias, Amanda L; Murray, John Isaac (2016) Combinatorial decoding of the invariant C. elegans embryonic lineage in space and time. Genesis 54:182-97|
|Walton, Travis; Preston, Elicia; Nair, Gautham et al. (2015) The Bicoid class homeodomain factors ceh-36/OTX and unc-30/PITX cooperate in C. elegans embryonic progenitor cells to regulate robust development. PLoS Genet 11:e1005003|
|Zacharias, Amanda L; Walton, Travis; Preston, Elicia et al. (2015) Quantitative Differences in Nuclear Î²-catenin and TCF Pattern Embryonic Cells in C. elegans. PLoS Genet 11:e1005585|
|Gage, Philip J; Kuang, Chen; Zacharias, Amanda L (2014) The homeodomain transcription factor PITX2 is required for specifying correct cell fates and establishing angiogenic privilege in the developing cornea. Dev Dyn 243:1391-400|