Mechanisms of Homeodomain Transcription Factor Specificity - PROGRAM SUMMARY/ABSTRACT: The goal of my research program is to determine how homeodomain (HD) transcription factors (TFs) that bind highly similar DNA sequences in vitro specify different developmental outcomes. HD genes constitute one of the largest groups of TFs in humans with over 200 family members that regulate a wide variety of processes including anterior-posterior patterning, organogenesis, and cell fate specification. Highlighting their impact on health, HD genes encode the most disease-associated variants in the Human Gene Mutation Database in comparison to other TF families. Collectively, over 100 HD genes have variants associated with diseases that impact numerous organs and tissues. Unfortunately, the identification of disease variants has greatly outpaced our understanding of their functional impact as hundreds of HD missense alleles have been classified as variants of unknown significance. Thus, it is imperative to define the underlying mechanisms to elucidate rules governing human variation and disease. While biochemical and structural studies have provided a wealth of information about how the HD binds DNA, recent studies revealed many variants cause unexpected changes in DNA binding. These findings expose three critical knowledge gaps: (1) How do HD proteins with highly similar DNA binding domains increase their DNA binding specificity to accurately regulate target genes? (2) How do the hundreds of HD missense variants associated with disease alter HD function? (3) Once bound to DNA, how do HD TFs activate and repress target genes and what role does the type of binding site play in transcriptional outcomes? To address these questions, I have assembled a transdisciplinary research team with complementary expertise that includes biochemical, bioinformatics, structural, cellular, genetic, and genomic approaches. Our team has recently developed a combined computational and experimental approach to discover that subsets of HD TFs form cooperative complexes on composite DNA sites with distinct site spacing and orientation requirements. Over the next five years, our plan is to use an iterative approach of using computational approaches for composite motif discovery followed by molecular, biochemical, and structural approaches to define the mechanisms underlying each cooperative complex. Comparative studies between different HDs and disease variants on different site configurations will provide novel insights into how HD factors gain DNA binding specificity and how disease variants cause molecular defects that lead to pathogenesis. We will develop new research tools and applications of high-throughput (HT) DNA binding assays, massively parallel reporter assays, and proteomic approaches to provide a comprehensive understanding of how binding affinity and cooperativity impact gene regulatory outcomes and define the co-factors used by different HD proteins to mediate accurate outcomes. Lastly, we will develop Drosophila and human organoid models that have unique strengths in genetics and genomics, respectively, to study the DNA binding and transcriptional regulatory potential of HD factors and analogous disease-associated variants within relevant developmental cell types.
