PROJECT SUMMARY The genomes of higher organisms are highly annotated by specific chromosomal proteins and histone modifications along active genes, regulatory elements, or silent regions. This annotation is critical for proper cell type specification, and an ongoing challenge is to decipher the rules that establish and maintain chromatin organization. My laboratory focuses on analysis of chromatin regulatory complexes, based on their central importance in development and disease, the intriguing hypotheses raised by our recent studies, and our ability to use new approaches to probe chromatin protein interactions with precision. Historically, we have made significant contributions to understanding the targeting and spreading of chromatin domains, and currently we are probing the ability of chromatin factors to poise genes for key regulatory decisions that specify cell type. Our current studies focus on the Polycomb group (PcG) proteins, and due to the high conservation of this key regulatory system we move between fly embryos and human embryonic stem cells with ease. We are developing a model in which key regulatory genes are universally ‘poised’ early in development via occupancy of composite protein complexes of Polycomb Repressive Complex 1 (PRC1) and classical co-activators. These ‘bivalent’ protein complexes may resolve into full activation or repression, depending on the cell type-specific expression, binding, and function of transcription factors. We speculate that transcription factors may bind relatively promiscuously, but still execute precise regulatory decisions, when they influence the local acetylation/deacetylation state at these predetermined sites. Our speculative model is based on strong proteomic evidence that PRC1 strongly interacts with classic co-activators, dBRD4 and dMOZ/MORF, captured on chromatin during embryogenesis in Drosophila, and by analogous co-occupancy of CBX7, RING2 (subunits of PRC1), and BRD1 (a subunit of MOZ/MORF) in human embryonic stem cells. We believe that we are on the cusp of understanding chromatin transitions and transcriptional programming at a mechanistic level. These studies also synergize with our molecular dissection of aberrant chromatin complexes that drive human cancers.