TITLE CasCUT&RUN: An in vivo method to analyze locus-specific protein complexes driving transcription of target genes in cancer Project Summary/Abstract Aberrant regulation of gene transcription commonly causes disease, including cancer. Transcriptional regulatory factors (TFs) are central players, binding to specific genomic response elements and nucleating assembly of multiprotein transcriptional regulatory complexes (TRCs) whose compositions and conformations are sensitive to gene, cell, and physiological context. For the many cancers in which a causative gene displays altered transcription, detailed analysis of causative TRCs would open direct routes to mechanisms (even if the driver is upstream, e.g., in a signaling pathway), and to potential treatments. However, no existing method can identify the unique combination of TFs and coregulator factors that occupy a single-locus mammalian response element in vivo. Described here are the development and validation of a new technology, CasCUT&RUN, which exploits at two steps the precision of Cas9 RNP genomic locus specificity to enable for the first time the isolation, purification and compositional identification of in vivo assembled, response element-specific TRCs from single loci in the human genome. The method will be unbiased, enabling identification of unique combinations of ~102 polypeptides that comprise individual TRCs, and amenable to future structural analysis by cryo-EM to detect conformational changes associated with altered regulation. Finally, CasCUT&RUN will be seamlessly adaptable to primary normal and tumor patient samples. Three specific aims are envisioned: 1. Develop and optimize: accuracy and sensitivity of isolation of promoter-bound RNA polymerase II transcription initiation complexes. Develop CasCUT&RUN using a collection of cell lines containing 1-200 copies of a single RNA polymerase II promoter, focused initially on recovering the many well-established promoter-bound proteins, and later on optimization to single copy sensitivity. 2. Isolate single locus TRCs and identify bound proteins in established cell lines. Use the same cell line collection to purify TRCs by CasCUT&RUN, focusing on the 1-200 copies of a glucocorticoid response element that confers hormone-inducible transcription on each of the linked promoters. When single copy sensitivity is achieved, isolate and analyze an endogenous single copy TRC in a second cell culture line. 3. Isolate single locus TRCs and identify bound proteins in primary cancer patient samples. Further develop CasCUT&RUN techniques for use in primary patient leukemia cells and formalin-fixed paraffin embedded solid tumor biopsies, and validate single copy TRC examined in Aim 2. These experiments will provide proof-of-principle for a new technology that can be applied to the wide range of cancers that display dysregulation of transcription of causative genes. CasCUT&RUN will also correlate structure, mechanism and pathophysiology in ways that could yield deep insight into combinatorial transcriptional regulation and its linkage to signaling networks, as well as pathways that produce or enhance cancer, or that cause resistance to therapies.