PROJECT SUMMARY Sequence-specific DNA binding by transcription factors (TFs) regulates RNA synthesis at target genes. This is a highly dynamic and context-dependent process, allowing cells to accurately regulate gene expression in response to diverse signals. While prior work has identified DNA binding preferences for numerous TFs, our understanding of the fundamental principles governing TF-DNA binding specificity and affinity remains incomplete. This is primarily due to limitations of current high-throughput methods, which fail to reliably detect low- to medium-affinity TF-DNA interactions that are more common and important for genomic binding in vivo. To address this gap, I have developed PADIT-seq, an innovative high-throughput reporter assay that enables functional testing and quantification of TF binding affinity across thousands of DNA sequences in a single experiment. The overall goal of this proposal is to utilize and further develop PADIT-seq to uncover fundamental TF-DNA binding principles, which will significantly advance our mechanistic understanding of the regulatory genome. Aim 1 will determine the role of medium-low affinity auxiliary binding sites and DNA shape features in determining TF genomic occupancy. I hypothesize that preferential recognition of sequential low-medium affinity sites flanking a core motif cooperatively increases residence time beyond what is predicted by core motifs alone. Aim 2 will apply PADIT-seq to identify the TFs whose binding is affected by pancreatic disease associated noncoding variants. It will also further develop PADIT-seq to make the assay even higher-throughput, enabling testing of higher numbers of TFs more easily. Finally, Aim 3 will elucidate intrinsic orientation biases of DNA binding domains in positioning partner proteins on DNA. Successful execution of these aims will provide major insights into the grammar and mechanisms underlying TF regulatory specificity. This work will also reveal how alterations in gene regulation driven by noncoding variants contribute to complex human diseases. My PhD research focused on identifying disease-associated genetic variants that are functionally relevant through genomic approaches. However, determining the precise TFs whose DNA binding is disrupted by these variants remained challenging. To overcome this barrier, my postdoctoral research has centered on pioneering a high-throughput PADIT-seq technology to sensitively profile how noncoding genetic variants alter TF-DNA interactions. While developing PADIT-seq has been a crucial advance, I need further training to complement this breakthrough with additional skills necessary to fully connect genotypes to cellular phenotypes. This will enable me to provide a complete picture elucidating how noncoding variants dysregulate transcriptional programs to promote disease. The K99/R00 award will provide invaluable training to equip me with the scientific and professional expertise needed to elucidate how noncod...