Testing the p53 code for Gene Regulation with Chemical Protein Synthesis

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With joint support from the Chemistry of Life Processes program in the Chemistry Division and the Genetic Mechanisms cluster in the Molecular and Cellular Biosciences Division, Dr. Champak Chatterjee from the University of Washington will investigate how modifications to the protein p53 control how human genes are turned on or off. The p53 protein controls how the genetic information in DNA is rewritten or transcribed to the intermediate message in the form of m-RNA. Mutations in the p53 transcription factor are known to cause human cancers. The ability of p53 to control genes is itself controlled by the reversible addition of other small proteins to this transcription factor. This project will use new synthetic chemistry to reversibly add variants of the protein known as a small ubiquitin-related modifier (SUMO) at specific positions of p53. Studies are designed to reveal how the SUMO modifications affect the ability of p53 to regulate the expression of human genes. This project will train graduate and undergraduate students in protein synthesis techniques, along with biophysical and biochemical assays of p53 function. In addition, an outreach program will introduce high school students to genetic disorders arising from mutations in transcription factors and provide research training to high school teachers. This project will construct specific post-translationally modified p53 proteins and use advanced biophysical and biochemical techniques to quantitatively characterize the roles of these modifications on the regulatory function of this transcription factor. Covalent conjugation with isoforms of SUMO and their acetylated forms on the disordered C-terminal region of p53 are known to regulate gene expression. This project provides a split-intein-mediated semisynthetic strategy to construct p53 modified by distinct SUMO isoforms, which are not readily available from biological approaches. Isoform-specific effects of p53 sumoylation that are confoundingly associated with both transcription activation and repression in cells will be carefully investigated through biochemical assays. Fundamental knowledge generated by this study will likely provide new insights into regulation of the master gene regulator in humans by reversible post-translational modifications.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.