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 <i>MYC</i> promoter and characterize the molecular effects of the bromodomain inhibitor JQ1, which targets bromodomain and extra-terminal (BET) proteins that regulate <i>MYC</i> expression. We quantified the consequences of BET protein displacement from the <i>MYC</i> promoter and found that it was associated with a considerable reorganization of the chromatin composition. Additionally, BET protein retention at the <i>MYC</i> 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.