Project summary There is a fundamental gap in our ability to target the non-coding genome for therapeutic benefit. While the druggable genome is expanding, it still remains confined to the tiny fraction of the human genome encoding for genes. This exclusion of the non-coding genome is unfortunate, as these regions can provide a powerful han- dle to finely control cellular responses and are more flexible than gene-targeting approaches. Our long-term goal is to develop a predictive and functional understanding of the non-coding genome, which will elucidate how these regions can be specifically targeted for genomic medicine. Towards this long-term goal, the objec- tive of this proposal is to systematically interrogate the combinatorial biology of enhancer gene regulation. We will pursue this objective at three levels: from the action of multiple protein effectors on individual enhancers, to the cooperativity between multiple enhancers, and culminating in the genome-wide coordination of multiple transcription factors to influence cell state. We have developed an innovative platform that enables high throughput assessment of combinatorial gene regulation, which will play a central role in this proposal. Our overall hypothesis is that, across multiple levels, combinatorial gene regulation uses a small set of components to create a broad spectrum of functionalities. We further hypothesize that the rules governing these functionali- ties can be explained by defining the key components, and examining how each functions alone and in simple combinations. The rationale for the proposed research is that a fundamental understanding of combinatorial gene regulation will enable predictive targeting of the non-coding genome for genomic medicine, leading to in- novative approaches to the prevention and treatment of disease. This proposal opens new research horizons to understanding the underlying concepts of combinatorial gene regulation and the epigenetic mechanisms un- derlying enhancer function, and for improved methods to modulate enhancer activity and engineer custom gene regulatory programs. Ultimately, this knowledge will be required to harness the genome for engineering and to understand how non-coding regulators contribute to development and disease.