TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome

Published: 13 November 2024| Version 2 | DOI: 10.17632/j58yghngky.2
Contributor:
Bercin Cenik

Description

Regulation of gene expression during development and stress response requires the concerted action of transcription factors and chromatin-binding proteins. Because this process is cell type-specific and varies with cellular conditions, mapping of chromatin factors at individual regulatory loci is crucial for understanding cis-regulatory control. Previous methods only characterize static protein binding. We present TurboCas, a method combining a proximity labeling enzyme miniTurbo with CRISPR/dCas9 that allows for efficient and site-specific labeling of chromatin factors in mammalian cells. Validating TurboCas at the FOS promoter, we identify proteins recruited upon heat shock, cross-validated via RNA polymerase II and P-TEFb immunoprecipitation. These methodologies reveal canonical and uncharacterized factors that function to activate heat shock-responsive genes. Applying TurboCas to the MYC promoter, we identify two P-TEFb coactivators, Super Elongation Complex and BRD4, as MYC regulators. TurboCas significantly improves locus-targeted proximity labeling, with potential to deepen understanding of regulatory pathways in development and stress response. These are uncropped and/or unprocessed imaging files from this study.

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Northwestern University

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Western Blot

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