Coupling Proximity Biotinylation with Genomic Targeting to Characterize Locus-Specific Changes in Chromatin Environments

Regulating gene expression
involves significant changes
in the
chromatin environment at the locus level, especially at regulatory
sequences. However, their modulation following pharmacological treatments
or pathological conditions remain mostly undetermined. Here, we report
versatile locus-specific proteomics tools to address this knowledge
gap, which combine the targeting ability of the CRISPR/Cas9 system
and the protein-labeling capability of the highly reactive biotin
ligases TurboID (in CasTurbo) and UltraID (in CasUltra). CasTurbo
and CasUltra enabled rapid chromatin protein labeling at repetitive
sequences like centromeres and telomeres, as well as nonamplified
genes. We applied CasUltra to A375 melanoma cell lines to decipher
the protein environment of the MYC promoter and characterize
the molecular effects of the bromodomain inhibitor JQ1, which targets
bromodomain and extra-terminal (BET) proteins that regulate MYC expression. We quantified the consequences of BET protein
displacement from the MYC promoter and found that
it was associated with a considerable reorganization of the chromatin
composition. Additionally, BET protein retention at the MYC promoter was consistent with a model of increased JQ1 resistance.
Thus, through the combination of proximity biotinylation and CRISPR/Cas9
genomic targeting, CasTurbo and CasUltra have successfully demonstrated
their utility in profiling the proteome associated with a genomic
locus in living cells.