We have recently developed a genetic screening approach, termed E-MAP (Epistatic MiniArray Technology Profiling), that can quantify the strength of systematically generated pair-wise genetic interactions. The method identifies negative double mutant interactions, where combination of mutations causes defects leads to enhancement of growth defects or lethality. Such interactions often specify membership of parallel biochemical pathways. Additionally, E-MAP also identifies positive interactions, where combination of mutants show mutual suppression or lack any additive defects, which are enriched among physically interacting gene products. Using gene deletions of non-essential genes and hypomorphic alleles of essential genes, we have recently generated E-MAPs in S. cerevisiae that have focused on 1) the early secretory pathway and 2) chromosome biology, which includes transcriptional regulation, chromatin remodeling and DNA repair. We now propose the second generation of E-MAP analysis, allowing us to address the next level of complexity, via examination of point mutants of multifunctional and essential genes. Specifically, we will genetically dissect two essential, multisubunit, multifunctional complexes at the heart of gene expression and chromatin structure: RNA polymerase II (RNAPII) and the nucleosome. This approach will allow us to A) map the structural features of these complexes onto their functional roles, and B) characterize the functional relationships between RNAPII and the nucleosome and the wider gene expression apparatus. In Aim #1, we will use the chromosome biology E-MAP to genetically examine a set of approximately 450 histone H3 and H4 mutants including A) complete alanine (or serine)-scans, B) comprehensive substitution of modifiable residues, and C) semi- systematic deletions of the N-terminal tails. The work, which is being done collaboratively with NIH Roadmap TCNP (Technology Center for Networks and Pathways) of Lysine Modification (PI Jef Boeke), will help reveal how histone-histone and histone-DNA contacts and histone modifications influence the steps of transcription and chromatin regulation. In Aim #2, we will screen approximately 100 distinct and diverse point mutants of several essential RNAPII subunits isolated in collaboration with Craig Kaplan and Roger Kornberg. In Aim #3, we will subject these data to hierarchical clustering and our recently developed metrics (S- and COP-scoring systems) to help identify functional relationships using the E-MAP data. We will also employ newly developed algorithms that identify functionally related sets of genes (or modules) from large-scale interaction datasets and allows for multi-functional genes to be members of more than one module. We anticipate that a systematic genetic approach described here will provide a more holistic view of chromatin function and transcriptional regulation in eukaryotic cells. The interplay between the gene expression machinery and the compaction of genetic material into chromatin is now recognized as highly dynamic and complex. These processes must be tightly controlled for normal cellular physiology and to prevent disease states such as cancer. We will use a systems approach to genetically dissect the structural and functional relationships between the highly conserved components of these pathways in the budding yeast, S. cerevisiae, in order to more easily understand and eventually manipulate the vast complexity of human cells.