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

ABSTRACT: The regulation of gene expression is a complex process that requires the concerted action of transcription factors and chromatin binding proteins. Because this process is both unique to a given locus and varied in response to changing cellular conditions, dynamically mapping the chromatin binding activity at individual promoters and other regulatory loci is crucial to understanding how cis-regulatory elements control gene expression. Earlier methods could only characterize the static binding activity of a single given protein. However, the recent emergence of proximity labeling, a technique for interrogating protein-protein interactions, and the advances brought about by CRISPR technology have enabled the development of new methods that can dynamically map the chromatin binding and secondary association of multiple proteins at given loci. In this study we describe TurboCas, a method that leverages the latest generation of proximity labeling enzymes, miniTurbo, in combination with catalytically dead Cas9 to label chromatin-binding proteins efficiently, dynamically and in a site-specific manner. We demonstrate the use of TurboCas to identify proteins binding the promoter of the heat shock responsive FOS gene. At the FOS promoter, we identify both canonical regulators, such as HSPA8 and members of the HSP network; and members of novel pathways, including the Hippo pathway, fatty acid synthesis pathways and the SUMOylation pathway. TurboCas represents a significant improvement over previous locus-targeted proximity labelling methods, with the potential not only to deepen our understanding of regulatory pathways in cellular stress response, but more broadly to advance the transcriptional regulation and chromatin biology fields.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Bercin Cenik  

PROVIDER: MSV000092561 | MassIVE | Mon Jul 31 11:54:00 BST 2023

SECONDARY ACCESSION(S): PXD044237

REPOSITORIES: MassIVE