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Existing methods for studying chromatin-binding proteins have significant limitations. These approaches preclude dynamic mapping of factor activity at specific genomic regions.
Cenik et al. developed TurboCas, a novel method combining proximity labeling (PL) enzyme miniTurbo with catalytically dead CRISPR-Cas9 (dCas9) to enable efficient, site-specific labeling of chromatin factors in mammalian cells. By pairing dCas9 with a single-guide RNA (sgRNA) and the miniTurbo enzyme, the researchers out of the Shilatifard lab can constrain protein labeling to a small radius at specific genomic loci.
The method was validated by investigating protein interactions at the FOS gene promoter during heat shock and the MYC locus. Using mass spectrometry, the researchers identified both known and novel regulators, including FUBP3 – a protein crucial for heat shock transcriptional response – and CDK9-containing complexes like BRD4 and the super elongation complex. A total of 16,559 peptides and 3,304 proteins were identified across the 3 replicates, including identification of 110 significantly enriched proteins (shared between 2 MYC guide RNAs).
Mass spectrometry was performed using a Vanquish Neo nano-LC coupled to an Orbitrap Exploris 240 hybrid quadrupole-orbitrap mass spectrometer. The analytical separation used an IonOpticks Aurora Elite CSI 15×75 C18 UHPLC column, connected to the mass spec with a Nanospray Flex ion source, with peptides eluted using an increasing organic gradient.
TurboCas advances the study of protein dynamics, offering precise temporal control and the ability to map protein interactions under varying cellular conditions. This technique could help understand transcriptional regulation in development and stress responses.
Publication
Molecular Cell
Authors
Bercin K. Cenik, Yuki Aoi, Marta Iwanaszko, Benjamin C. Howard, Marc A. Morgan, Grant D. Andersen, Elizabeth T. Bartom, & Ali Shilatifard
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