Project Summary The human genome exists in the cell nucleus as chromatin, a complex of the DNA with histone proteins. Though genetic information is encoded in the DNA sequence, another layer of information, is encoded in the histone proteins, specifically in the form of post-translational modifications (PTMs). This layer of information is often referred to as epigenomics, and provides instructions on how the genome is to be regulated. Chromatin and the epigenomic content, is highly dynamic, constantly restructuring in response to developmental and environmental cues. One of the most important questions in biology is how this information is interpreted by transcriptional and other regulatory complexes, leading to gene regulation and cell fate. Histone modifications are “read” through small subdomains within the regulatory complexes called reader domains, and specificity for a unique epigenomic pattern is thought to be achieved through the integrated activity of multiples of these reader domains. However, though much is known about the association of reader domains with fragments of histones, the molecular mechanims underlying how they associate with histones in a chromatin relevent context, or how they function together to readout a specific epigenomic state, are not well understood. This research program addresses this fundamental question in chromatin regulation. We are pioneering the use of NMR spectroscopy to study the association of reader domains with the basic unit of chromatin, the nucleosome. We are combining this with fluorescence microscopy, cryo-electron microscopy, and basic biochemistry for an overall multidisciplinary approach to building models of these complexes. Over the next five years we will focus on how the conformation of the nucleosome regulates readout of epigenomic signatures. We will continue our investigation of the nucleosome conformation itself and how known cancer mutations dysregulate this. In addition, we will determine the kinetic and structural basis of association of reader domains from the PBAF chromatin remodeling and PRC1 histone modifying complexes with nucleosomes. Through collaborative studies we will investigate the functional consequence of these interactions. We will continue to build towards our long-term goal of understand of how multiple reader domains integrate to allow regulatory complexes to navigate and respond to a dynamic chromatin substrate. Results from this research program will reveal fundamental mechanisms of chromatin regulation, provide insight into the etiology of a number of human diseases, and lay the groundwork for the development of targeted therapeutics.