Title: Death-Seq, a Method for Genome-wide Identification of Functional Silencer Elements Summary The human genome contains an estimated 98.5% of non-coding DNA 1. Little is known about the function of this non-coding DNA, and limited tools are available to assay for functional non-coding regions. These non-coding regions contain essential regulatory elements (e.g., enhancers, silencers, insulators) that are paramount during normal human development and whose dysregulation is implicated in numerous human diseases. Mapping regulatory elements in our genome currently relies upon structural assays, location of transcription factor binding sites or chromatin marks and a few functional assays, including STARR-Seq (a self-transcribing reporter assay to measure enhancer functionality). Despite increasing knowledge of the function and location of enhancers, no tool exists to functionally assay silencer elements. We propose a novel method, termed Death-Seq, to interrogate genome-wide DNA for functional silencer ability by negative selection. Briefly, genomic libraries for interrogation will regulate expression of a suicide gene within a transfectable vector. Under the control of an enhancer element, the suicide gene will express and induce cell death, leading to vector depletion. A silencer element will repress suicide gene expression, leading to cell (and vector) survival. After selection, plasmids from the surviving cells will be sequenced and are expected to contain only functional silencer elements. Aim 1 proposes to identify constitutive silencers using a Caspase 9- based suicide gene to induce apoptosis. Aim 2 describes a variation the approach, allowing for silencer interrogation at user-defined timepoints (for detection of silencers dependent on inducible repressors). This tool addresses an unmet need in the gene regulation community, as no current technique allows for genome-wide study of silencer function. Additionally, Death-Seq may be used in future investigations to test diverse hypotheses; its versatility allows for interrogation of varied input libraries (e.g., genomic or ATAC/ChIP-Seq), and its transfectability allows for study of silencers in diverse cell types. Death-Seq will allow for the cataloguing of genomic silencer elements, both cell-type specific and universal. The location of a functional silencer will enhance understanding of transcriptional and chromatin regulation by transcription factors. Identifying novel silencer motifs, as well as genome-wide characteristics of silencer locations, will benefit the community of genomic research broadly. Identifying silencer element locations in non- coding results of genome-wide association studies (GWAS) will yield directions for future rational research into the mechanisms of human disease. We believe that the tool developed through this proposal, Death-Seq, will provide an innovative yet feasible solution to investigating silencer biology.