Project Summary Cells sharing a common genome transcribe a subset of their available genes to perform different functions. Polycomb-group (PcG) proteins contribute to cell specialization by binding and chemically modifying chromatin around lowly-transcribed genes to further reduce (“silence”) their expression. Although many PcG proteins have been identified, mystery surrounds the most interesting aspects of their function. The utility of silencing derives from its selective imposition. However, unknown mechanisms determine when silencing begins or ends during normal development. Furthermore, how PcG proteins epigenetically associate with different target genes in different cells is poorly understood. It is important to resolve these questions because silencing mistakes disrupt development and contribute to human cancers. The proposed research program uses a new model system, Drosophila nurse cell differentiation, and new genetic and genomic engineering tools to study how silencing initiation, maintenance, and reversal are developmentally regulated. The proposed model system overcomes technical limitations of studying silencing initiation in early embryos and embryonic cell cultures. A new proposed tool, termed UAS-gene swap, allows genetic substitution of PcG proteins with mutant versions in thousands of cells, and is compatible with sensitive molecular approaches like ChIPseq. The first proposed research direction will use UAS-gene swap to identify the interactions between PcG proteins and transcription factors that are important for establishing or preventing silencing at each potential Polycomb target gene. A second research direction will explore how a single PcG protein, Scm, is post-translationally regulated to time silencing initiation in differentiating nurse cells and other developmental contexts. Finally, a third research direction will use a highly- efficient genome engineering approach to explore how some Polycomb target genes are stably silenced while others are not. These diverse directions will significantly advance our understanding of how development regulates Polycomb silencing. They will also generate new tools to study gene expression control in developing organisms that will be valuable to the broader research community. Lastly, this research program may reveal potentially conserved mechanisms that regulate early embryonic development and prevent human cancers.