Project Summary/Abstract Gene regulation is central to all life, normal and diseased. The long-term goal of this research program is to un- derstand the molecular mechanisms governing the regulation of all genes in yeast and human systems. This basic knowledge will help produce better diagnostics and treatment options for people. Regulation of the hu- man genome is very complex. Therefore, this research program is focused initially on the simpler yeast Saccha- romyces to experimentally dissect mechanisms of gene regulation that are fundamental and common to all eu- karyotic life. Concepts developed in yeast are ultimately tested in human cells, thereby accelerating discovery. This research program has developed an ultra-high-resolution assay called ChIP-exo to map the bound locations of essentially any protein throughout any genome at base-pair resolution. Using this strategy, a com- prehensive first-of-its-kind epigenome map of the protein-DNA architecture of yeast cells was established. This is now being established in human cells. The epigenome is defined here as the compilation of all molecular in- teractions with DNA and RNA, beyond base-pairing. The next phase of this research is to understand the func- tional interactions among the protein components of the epigenome. This will be achieved in part through dele- tion, mutation, and/or rapid depletion of protein components of the epigenome, particularly those involved in inducible and constitutive transcription. The former is gene-specific and can be hyper-expressed in response to specific signaling events. The latter is general to most genes and typically occurs at low levels. Importantly, the research program here is defining the protein architecture that specifies inducible versus constitutive promot- ers. Once protein components of this architecture are experimentally removed (e.g., sequence-specific tran- scription factors or their cofactors), then the impact of this removal will be measured on chromatin organiza- tion, loading of the core transcription machinery and subsequent transcription. A parallel strategy will be em- ployed in human tissue culture cells to assess conserved paradigms. This research will also continue with its previous biochemical reconstitution of chromatin organization across entire genomes using purified proteins, but now adding in components of the transcription machinery and their regulatory factors. A biochemical system will provide greater control over the experimental parame- ters and therefore provide greater insight into molecular mechanisms of gene control. It is now clear that in- duced genes coalesce in 3D space within the nucleus. However, it remains unclear which genes coalesce into which hubs. Therefore, 3D mapping technologies like SPRITE will be adopted to measure gene clustering. This will provide insight into how multiple genes become coordinately induced by regulatory signals. Taken to- gether, the product of this research program will be a d...