CasCUT&RUN: An in vivo method to analyze locus-specific protein complexes driving transcription of target genes in cancer


Year of Award:
2019
Status:
Active
Award Type:
R21
Project Number:
CA235303
RFA Number:
RFA-CA-18-002
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
YAMAMOTO, KEITH ROBERT
Other PI or Project Leader:
KROGAN, NEVAN J
Institution:
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
TITLECasCUT&RUN: An in vivo method to analyze locus-specific protein complexes drivingtranscription of target genes in cancerProject Summary/Abstract Aberrant regulation of gene transcription commonly causes disease, including cancer. Transcriptionalregulatory factors (TFs) are central players, binding to specific genomic response elements and nucleatingassembly of multiprotein transcriptional regulatory complexes (TRCs) whose compositions and conformationsare sensitive to gene, cell, and physiological context. For the many cancers in which a causative gene displaysaltered transcription, detailed analysis of causative TRCs would open direct routes to mechanisms (even if thedriver is upstream, e.g., in a signaling pathway), and to potential treatments. However, no existing method canidentify the unique combination of TFs and coregulator factors that occupy a single-locus mammalian responseelement in vivo. Described here are the development and validation of a new technology, CasCUT&RUN, whichexploits 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 singleloci in the human genome. The method will be unbiased, enabling identification of unique combinations of ~102polypeptides that comprise individual TRCs, and amenable to future structural analysis by cryo-EM to detectconformational changes associated with altered regulation. Finally, CasCUT&RUN will be seamlessly adaptableto 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 IItranscription initiation complexes. Develop CasCUT&RUN using a collection of cell lines containing 1-200copies of a single RNA polymerase II promoter, focused initially on recovering the many well-establishedpromoter-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 linecollection to purify TRCs by CasCUT&RUN, focusing on the 1-200 copies of a glucocorticoid response elementthat confers hormone-inducible transcription on each of the linked promoters. When single copy sensitivity isachieved, 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. Furtherdevelop CasCUT&RUN techniques for use in primary patient leukemia cells and formalin-fixed paraffinembedded 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 rangeof cancers that display dysregulation of transcription of causative genes. CasCUT&RUN will also correlatestructure, mechanism and pathophysiology in ways that could yield deep insight into combinatorialtranscriptional regulation and its linkage to signaling networks, as well as pathways that produce or enhancecancer, or that cause resistance to therapies.