Polycomb group (PcG) and Trithorax group (TrxG) proteins are essential for maintaining different cell identities through chromatin-based regulation of gene expression during development. A crucial aspect of this system is the competition of PcG and TrxG proteins for cis-regulatory DNA elements called Polycomb Response Elements (PREs). However, the mechanisms underlying this competition remain largely unknown. Recent studies have shown that PREs are nucleosome-depleted sites, as a result of high nucleosome turnover in Drosophila. In addition, our preliminary results suggest that these transient nucleosomes at PREs are required for Polycomb-mediated repression. Here, we propose to test the idea that PRE chromatin structure dynamically alternates between two states, where PcG proteins initially bind to transient PRE nucleosomes, and TrxG proteins displace these nucleosomes allowing access to underlying binding sites in the DNA. First, we will evaluate the relevance of transient PRE nucleosomes for establishment and maintenance of target gene expression patterning in vivo. Second, we will implement an innovative technique for genome-scale epigenomic profiling, based on Micrococcal nuclease digestion and high-throughput DNA sequencing. Our recent work shows that this method reveals every site that is occupied by DNA-binding proteins and nucleosomes. We will use this technique in wild-type and nucleosome-depleted cells to assess the roles of transient nucleosomes for chromatin organization at PREs. Finally, we will directly test the role for transient nucleosomes in the recruitment of PcG/TrxG complexes at PREs by chromatin immunoprecipitation analysis. This project will explore links between nucleosome turnover and PRE function, while achieving significant technological innovation. The outgoing phase will be hosted by Dr. Kami Ahmad at Harvard Medical School, in Boston, while Dr. Geneviève Almouzni at the Curie Institute, in Paris will be the return host.
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