Engineering Mice and Systems for Analysis of Chromatin Regulation: CiAO and FIRE-Cas9 systems

During recent years, genomic analysis has shown that mutations in chromatin regulators contribute to about 50% of all human malignancies and a substantial fraction of human development diseases. Despite the importance of chromatin regulation, mechanistic analysis that might lead to therapeutic developments has been difficult. In the past chromatin regulation has been largely studied by traditional biochemical in vitro approaches involving transcription on nucleosomal templates. However, these approaches cannot reconstitute some of the most interesting and important aspects of chromatin regulation such as topologic actions, long range regulatory effects or the functions of highly tissue specific chromatin modifications. To overcome these difficulties we have been developing approaches that use chemical induced proximity (CIP) (Stanton, BZ, Chory, EJ and GR Crabtree, Science in press) and genetically engineered mice to study chromatin regulation in vivo. These approaches let the cell form chromatin in all its topological and cell specific complexity and then use CIP to introduce a specific chromatin regulator to a specific genetic locus. The orderly sequence of biochemical events are then followed allowing one to mathematically model the kinetics of the actions of single chromatin regulator executing its function within a characterized chromatin environment (Hathaway et al Cell 2012; Hodges and Crabtree PNAS 2013: Kadoch C et al Nature Genetics 2017; Stanton et al Nature Genetics 2017, Miller, E. et al Nature Molecular and Structural Biology 2017). Recently we have developed a CRISP based method (FIRE-CAS9) that allows one to examine the mechanism of action of a single chromatin regulator at any genetic locus in nearly any cell type (Braun S et al Nature Comm. 2017). Because of the reversibility of recruitment with CIP, these approaches allow accurate modeling of chromatin memory and the analysis of mechanisms not possible with more traditional reconstitution approaches used in the past by ourselves and others. Use of these approaches have allowed us to discern the mechanism of opposition between BAF (a trithorax member and Polycomb) demonstrating that the mechanism underlying the critical opposition is a direct, ATP-dependent interaction between BAF and PRC1 resulting in PRC1 and PRC2 removal within minutes and the development of accessible DNA without transcription, PolII occupancy or cellular replication.