Summary
Embryonic stem cells (ESCs) posses an unlimited capacity to self-renew under defined culture conditions, while retaining the potential for multi-lineage differentiation. Understanding how these unique properties can be harnessed to generate specific progenitors and terminally differentiated cell types is of key interest to clinical applications. In addition, improved insight into the mechanisms regulating ESC identity is relevant to cancer research, because ESC self-renewal programs are often hijacked during the development of malignancies. Polycomb-repressive protein complexes (PRCs) are important regulators of ESC pluripotency implicated in tumorigenesis. Here, we propose to perform in-depth proteomic analyses of two distinct PRC complexes (PRC1 and PRC2) using a single-step purification technique based on specific in-vivo biotinylation combined with SILAC labeling and mass spectrometry. Although the core components of both PRCs are well defined, the precise complex composition is likely to vary during differentiation to allow the establishment of distinct patterns of gene expression. Therefore, we will analyze these dynamic changes in mouse ES cells (mESC) and neural precursors derived from these cells using a well-defined neural differentiation protocol. As phosphorylation controls PRC1 activity, we will also study specific phosphorylation events on PRC1 core members by a combination of stable isotopic labeling techniques with peptide spiking for accurate quantitation and SCX/TiO2 chromatography for efficient phosphopeptide enrichment. Finally, we will use synchronized cell cultures to map dynamic changes and phosphorylation states of PRC1 and PRC2 complexes during cell cycle progression. Together, these projects are expected to bring novel mechanistic insights in how Polycomb repressive complexes regulate cell fate decisions.