The Regulatory Consequences of Transcription Factor Evolution

Carnegie Mellon University, Pittsburgh PA

Investigators

Abstract

This project will explain how evolutionary changes in an animal's DNA sequence lead to evolution of morphology, and will more fully explain how genomes work. Genomes are broadly comprised of genes which encode proteins, and the non-gene, noncoding regions. An important function of these noncoding DNA sequences is to bind proteins, which in turn will control when, where, and to what level the genes are activated, or expressed. How these DNA:protein interactions evolve and direct changes in gene expression is almost entirely unknown. The research here will contribute to an understanding of how proteins can evolve changes in the strength, or affinity, of DNA binding, and how this leads to the evolution of gene expression. The project will also provide training opportunities to a postdoctoral fellow, including opportunities for training in mentoring more junior scientists. Undergraduates will be meaningfully engaged in hands-on, self-directed research training in genomics, genetics, bioinformatics and evolution. Such cross-disciplinary training is critical for preparing the next generation of biologists. The lab group will also reach out to local area high school students by providing a module in animal diversity, evolution, and the use of genome and bioinformatics tools, thereby extending the existing curriculum and engaging students early in their biology education. The sequencing data generated from this research will be housed in an online, openly accessible database. This project will provide the first comparative, broad-scale, in vivo experimental validation that low- and high-affinity transcription factor binding sites confer functional differences in gene expression and will contribute to an understanding of how this has evolved. Recent technologies have been developed that allow assessment of the range of DNA binding preferences and associated affinities for transcription factors. Use of these technologies demonstrated that three orthologous transcription factors--mouse Eomesodermin, sea urchin Tbrain and sea star Tbrain--have maintained almost identical high-affinity binding motif preferences but have evolved changed preferences for a low-affinity motif. The project here directly extends this work to examine the roles that transcription factor DNA binding affinity may have in regulating gene expression, and the consequences that this has for phenotype. Using echinoderm embryos as a model, the project uses whole-genome ChIP-Seq and RNA-Seq, together with a new, medium-throughput "nanotag" method, to assess regulatory function in vivo. The aim is to determine the association between motif use and response to changing levels of Tbrain and its putative cofactor Smad2/3. This will test whether high-affinity or low-affinity motifs are most responsive to changing levels of Tbr protein, if association depends on the number of sites, and how this response will differ between the two species. The work is expected to provide a new conceptual framework for how gene expression can evolve through use of high- and low-affinity binding sites.