PROBLEM: The protein interaction module called the Sterile Alpha Motif (SAM), which is found in many proteins including those involved in gene regulation, possesses the unusual ability to self-associate into an open-ended helical polymer architecture. Over a decade has passed since solving the first such SAM polymer structure suggested the intriguing possibility that these polymers could control chromatin architecture. In part due to technical barriers, key mechanistic questions regarding the function of SAM polymers have remained unanswered: 1) how are SAM polymers regulated? 2) how do SAM polymers control chromatin structure and influence gene expression? OBJECTIVE: This proposal focuses on a SAM-containing, chromatin-associated protein called Polyhomeotic (Ph). Ph is a member of the Polycomb group (PcG) of epigenetic regulatory proteins which play a critical role in development and have been implicated in cancer progression.
Our aims are: 1) dissect the mechanism and regulation of Ph SAM polymerization activity; 2) determine the composition and stoichiometry of Ph oligomers in cells; 3) determine how Ph SAM polymerization affects chromatin binding and organization. By utilizing new technologies, our goal is to finally understand how the Ph SAM polymer controls chromatin architecture and gene expression. METHODS: We will utilize a broad spectrum of approaches to understand Ph SAM polymers. First, we will use structure guided mutations and biophysical methods (including analytical ultracentrifugation) to determine the molecular basis by which Ph SAM polymerization is regulated by other sequences in the Ph protein. This analysis will be extended using super-resolution microscopy, in vitro reconstitution, and mass spectrometry to dissect how Ph SAM polymerization can occur in the multi-protein PcG complexes in which Ph exists in cells. How these SAM-dependent polymers affect chromatin structure will then be determined using a combination of in vitro reconstitution and high resolution analysis of chromatin in cells (using chromosome conformation capture and next generation sequencing, restriction enzyme accessibility assays, and chromatin immunoprecipitation). Finally, Ph mutants with disrupted or enhanced SAM polymerization activity will be tested for their effect on gene expression and cell growth in developing Drosophila and cell culture. SIGNIFICANCE: SAMs are found in many proteins spanning from chromatin regulators to membrane proteins. The conserved architecture of the SAM and possibly of the mechanisms that regulate it mean that our dissection of Ph SAM has general implications for SAM-containing proteins, and for possible therapeutic approaches based on modulating protein polymerization. Much of the work on PcG proteins and other chromatin regulators has focused on histone modifications. Our work on Ph SAM polymerization activity is revealing new mechanisms for regulating gene expression by controlling protein and chromatin architecture.
Numerous diseases such as cancer stem from the inability to properly organize genomic information into large molecular assemblies called chromatin. Our previous work published more than a decade ago, on a protein module called SAM, revealed an unexpected polymer structure which we proposed at the time was a novel way for regulating chromatin structure and consequently, gene expression. It is only now with the availability of new technologies that we can finally address the long standing question of how polymeric SAMs affect chromatin structure, and ultimately, provide the means to manipulate gene expression for therapeutic benefit.
|Wong, Sarah J; Gearhart, Micah D; Taylor, Alexander B et al. (2016) KDM2B Recruitment of the Polycomb Group Complex, PRC1.1, Requires Cooperation between PCGF1 and BCORL1. Structure 24:1795-1801|