Developing a multi-species platform for understanding mammalian gene control

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Transcription factors (TF) are proteins that bind to DNA and regulate the expression of genes. Compared to protein-coding gene mutations, relatively few functional regulatory mutations have been characterized. The overarching goal of this proposal is to identify and determine the function of evolutionarily conserved and species-specific DNA regions that interact with TFs and regulate genes in specific cell types. Recent technological advances allowing genome-wide experimental isolation of TF-bound DNA sequences have revealed that in any given cell type, there are more TF bound DNA segments than there are genes. Consequentially, this has created a great need for new ways to filter hundreds of thousands of TF-DNA interactions down to the most essential ones that impact gene function. Comparing DNA between species is a long-established way to identify gene function. Our recent discoveries, comparing TF-DNA interactions in liver tissue from 5 vertebrate species, revealed that conserved TF-DNA interactions significantly enriched for liver TF networks and important conserved liver pathways such as blood clotting factor production and lipid metabolism. There remains, however, a great need to develop a truly multi-species platform that captures normal biological processes, yet is still amenable to experimental manipulation. In this proposal, we will first create a new multi-species TF-DNA interaction atlas in endothelial cells isolated from several mammalian species, including human, dog, mouse, rat and cow. We will then test our prediction that conserved regulatory regions are the important functional ones by performing targeted regulatory DNA deletions. Overall, this work will allow us to make new discoveries regarding the evolution of mammalian gene control.