Tag-ChIP-MS for analysis of chromatin-level regulation of DNA repair


Year of Award:
2017
Award Type:
R21
Project Number:
CA213247-01
RFA Number:
RFA-CA-16-001
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
KRON, STEPHEN J
Other PI or Project Leader:
Not Applicable
Institution:
UNIVERSITY OF CHICAGO
Abstract Recent progress on proteomic analysis of chromatin has led to significant discoveries but has also revealed limitations of current ChIP-MS strategies. In particular, comparing results among experiments and laboratories is challenging. Here, we will leverage advanced tools for genome editing, protein tagging, chromatin enrichment and LC-MS/MS analysis to establish a new approach to chromatin proteomics, Tag- ChIP-MS, that also has significant potential for wider applications. Using the chromatin response to DNA double strand breaks as a model, we will examine the relocalization of proteins to DNA damage foci, isolate the proteins along with their chromatin context, and demonstrate capability to explore the proteins and modifications with high sensitivity, specificity and reproducibility. By establishing a facile means to insert imaging-and-capture tags on the chromosome, we will also provide a broadly useful tool for a wide range of cellular assays, including ChIP-seq, ChIP-MS and other studies of dynamic protein and protein/nucleic acid complexes in cells and animals.