PROBER identifies proteins associated with programmable sequence-specific DNA in living cells

DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells. PROBER offers a method to identify proteins associated with the DNA sequence of interest and quantify differential binding caused by sequence variants in living cells.

Automated summary

DNA-protein interactions play a crucial role in gene regulation and can be affected by DNA variants associated with polygenic diseases. A new method called PROBER has been developed to rapidly identify and quantify proteins associated with specific DNA sequences in living cells. PROBER utilizes high-copy episomes and proximity proteomics to accurately identify transcription factors and gene regulators associated with target DNA sequences and single-nucleotide variants. The method has been used to study the impact of cancer hotspot mutations on regulator associations.