Chemical strategies to investigate biochemical crosstalk in human chromatin

PROJECT SUMMARY Chromatin is a massive nucleoprotein complex that packages about three billion base pairs of genetic information in nucleated cells. Histones and transcription factors (TFs) are two important families of proteins associated with chromatin that play key roles in organizing, protecting and activating our genes. A conserved feature among histones and TFs is the critical role that post-translational modifications (PTMs) play in controlling their diverse functions. Many gaps remain in our understanding of the mechanistic roles for specific histone and TF PTMs that are either low in abundance or where the necessary molecular biological and chemical tools are unavailable for mechanistic studies. This is especially challenging when studying regulation of the tumor suppressor p53 that is rapidly turned over by the proteasomal machinery in our cells. The proposed research project seeks to overcome challenges arising from the low abundance and heterogeneity of histone and p53 modifications by applying a combination of chemical and molecular biological tools to generate site-specifically modified proteins. Specifically, sumoylated histones H2B and H4 and methylated p53 will be generated by new protein semisynthesis techniques. The semisynthetic proteins will be subjected to a range of biophysical and biochemical assays in order to elucidate the mechanistic roles for sumoylation and methylation in regulating chromatin structure and function. Results from biochemical assays will be further validated in cell- based studies to arrive at a complete picture of the molecular mechanisms underlying gene regulation by histone sumoylation and p53 methylation. The long-term goal of this project is to identify new biochemical relationships, or crosstalk, in cellular chromatin that may be controlled to engineer cellular fates and selectively therapeutically targeted in human diseases.