The segregation of genetic material into actively transcribed (euchromatin) and heritably repressed (heterochromatin) chromatin states is essential for the maintenance of cell identity. These chromatin states are maintained and rearranged by the combined action of ATP-dependent chromatin remodeling motors and specific chromatin binding proteins. The proposed work is aimed at studying the core mechanisms of these chromatin regulators to achieve a better understanding of how their activities are regulated in vivo. Using a variety of biophysical approaches we have found that chromatin remodeling ATPases take advantage of unexpected plasticity within the smallest unit of chromatin, a nucleosome. A plastic nucleosome suggests the presence of additional regulatory roles for chromatin. We have also uncovered phase-separation behavior in HP1 proteins, which are core components of heterochromatin. These results suggest that some of the repressive functions of heterochromatin may arise from physical sequestration of chromatin in phase-separated bodies. Here we will build on these new discoveries to ask the following questions: 1. How do chromatin remodeling motors couple ATP hydrolysis to changes in nucleosome conformation? 2. What are the differences in mechanism between remodelers from different classes? 3. What is the role of phase-separation in heterochromatin regulation?
Mis-regulation of chromatin states either via misregulation of ATP-dependent chromatin remodeling complexes such as ACF and SWI/SNF or via mis-regulation of HP1 mediated heterochromatin spread is strongly associated with cancer. For example, mutations in SWI/SNF subunits are thought to be driver mutations for several cancers and loss of gene silencing due to low levels of HP1 has been strongly linked to breast cancer metastasis. Yet how chromatin remodeling motors work or how heterochromatin is assembled and regulated is poorly understood. The proposal combines cutting edge biophysical approaches with in cell tests to tackle these challenging questions. We expect that our work will help identify key steps in chromatin remodeling reactions and heterochromatin assembly that can be targets for regulation and therapeutic intervention.
