Grant: Project Grant

Transcription factors are proteins that regulate gene expression, and play a pivotal role in normal development, homeostasis, and disease. Transcription factors work by binding to specific sequences within the DNA, altering expression of the adjacent genes. Mutations in transcription factors can alter the ways in which they bind DNA, changing which genes they regulate and how much those genes are expressed. Sometimes these mutations result in heritable predispositions to disease, and other times they occur within an individual and can lead to cancer. One of the most commonly mutated transcription factors in cancer is p53. Some p53 mutations appear to have rewired it to regulate a new set of genes, but little is known about the identities of the newly regulated genes or how this affects cancer because little is known about how mutant forms of p53 bind to DNA. We aim to build a platform for characterizing transcription factors in sufficient throughput that we can easily test how mutations in their protein sequence affect their ability to bind DNA for large numbers of mutations. First, we will demonstrate that our approach works by using it to characterize p53, a critical transcription factor in health and disease. Next, we will scale up this approach to characterize 96 transcription factors in a single experiment, and use the resulting data to determine how much more we can scale up this approach. Finally, we will create >1,000 mutant versions of p53, including many that result in heritable diseases and are commonly found in cancer to see how mutations alter p53 binding to DNA. This will demonstrate that our approach can be used to quantify the activity of mutant transcription factors in high throughput, enabling us to understand how mutations in transcription factors result in disease, and to design and test new transcription factors with desirable features.