Chromatin remodeler (CR) complexes play crucial roles in regulating chromosomal architectural and act to modify nucleosomal DNA contacts by repositioning nucleosomes. The mechanistic details of the CR family of proteins are of importance because each remodeler complex contributes to unique chromatin structural maintenance. A mechanistic understanding of these proteins requires resolving how they interact and influence the chromosomal fiber. CRs are comprised of an ATPase active subunit and numerous auxiliary components that are involved in elaborate protein-chromatin stabilizing interactions. This makes it a challenging task to resolve completely their structures and how each subunit may interact individually with the nucleosome. To date, there remains no high-resolution structural data for a remodeler complex bound to the nucleosome. A disadvantage to remodeler studies is that they often rely on the reconstitution of nucleosomal arrays in solution that cannot recapitulate the true nuclear environment. Therefore, to enhance the understanding of CR behavior a technique is required that illuminates the molecular contacts that occur between the nucleosome and CR proteins inside the living nucleus. This proposal will define an approach to resolve the molecular dynamics of CR proteins revealing a nucleosomal-CR interactome in living yeast. This will be achieved using a method that allows for the covalent trapping of histone-protein interactions in the nucleus by employing site-specific, UV-inducible crosslinker amino acids that are genetically incorporated into the nucleosome. Spatial details will be achieved in two ways. First, specific placement of the crosslinker within a histone protein will provide insights into the nucleosomal surface contacts made by CR subunits. Second, when this approach is paired with chromosome immunoprecipitation (CHiP) technologies it will be possible to determine the chromosomal positioning of the crosslinking event, detailing the occupancy of the interaction along the chromatin fiber. Temporal resolution will be obtained with the aid of synchronous cells and UV-control of the crosslinking event. Time-resolved in vivo crosslinking with genetically encoded UV-crosslinkers is ideally suited to address many of the open questions in chromosome structure, composition and formation. Most critically it has the potential to reveal the network of interactions within chromosomal pathways and identify the sequence of events involved in those mechanisms. Particularly, this approach will help define how histone posttranslational modification events influence CR binding and dynamics at the nucleosomal surface. This work will be the first to institute the technique to assess structural and dynamic crosslink mapping of chromosomal remodeler complexes, in vivo. This work will bridge an extensive gap that spans in vitro versus in vivo experimentation of CRs and assign biologically relevant structure/function dynamics to these large intricate complexes. The results will greatly advance the chromatin biology field. Furthermore, CRs are medicinal targets for disease and developmental disorders highlighting their relevance to public health. 1
Genetic integrity relies on the swift and faithful regulation of chromosomal restructuring that is controlled by the enzymatic actions of chromatin remodeler complexes. Misregulation of remodeler enzymes leads to inaccurate nucleosomal reorganization impairing proper chromosomal architecture that alters gene expression. Resolving the structural dynamics of these complexes, and how they physically interact with their targets, is of great importance to public health because errors in chromatin remodeling are the root of many human genetic disorders due to improper DNA translation, replication, and repair.