PROJECT SUMMARY Genome-wide molecular profiling of dynamic DNA and chromatin modifications across diverse cell and disease states have resulted in comprehensive catalogs of putative non-coding regulatory elements in the human genome. Perturbation experiments are now critical to learn the causal functions of the DNA & histone modifications at these genomic elements. However, existing tools to manipulate gene expression and chromatin state suffer from partial or transient effects, are large and thus difficult to deliver, and exhibit high variability across loci and cell types. These tools are currently drawn from a tiny fraction of the thousands of natural chromatin regulatory complexes. A more complete toolbox of compact, efficient domains capable of manipulating a broad range of chromatin pathways will transform our ability to determine the causal function of particular chromatin modifications across the human genome and to control gene expression. Here, we propose to systematically and comprehensively measure the gene expression effects of recruiting chromatin regulators and transcription factor protein domains that can interface with human chromatin - a critical missing dataset. This is made possible by our recent development of the first high-throughput protein domain recruitment assay in human cells, capable of measuring activity for tens of thousands of effector domains simultaneously (Tycko et al, bioRxiv 2020). Using this system, we will recruit-and-release effector domains from the promoter, wait, and then measure the magnitude and permanence of transcriptional silencing or activation at a reporter locus. We will characterize thousands of domains drawn from human and viral chromatin and gene regulators. In addition, we will create orthogonal nanobody libraries selected to recruit endogenous chromatin regulators. We will then measure the function of these domains at a panel of endogenous genomic loci chosen to represent diverse chromatin states. Finally, we will use genetic screens and epigenomic mapping assays to determine the molecular networks that underpin the functions of these novel effectors. Therefore, we will create and share a detailed resource of experimentally-measured, compact, efficient domains that can be fused onto DNA-binding proteins in order to recruit desired chromatin regulatory complexes to act upon a genomic element. At the same time, this study will provide detailed functional properties for all human transcriptional and chromatin regulatory domains, serving as a starting point for future work on understanding the disease implications of genetic mutations in the coding sequence of these regulators. Further, it will identify novel epigenome and transcriptional effector domains that can impart permanent epigenetic memory, and identify combinations of domains to impart a range of chromatin states not currently accessible with available perturbation tools. These pathway-specific perturbation technologies will be critic...