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

Smarajit Mondal1, Muthukumar Ramanathan2, Weili Miao1

  • 1Program in Epithelial Biology, Stanford University, Stanford, CA, USA.

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|August 4, 2022
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Summary

Researchers developed PROBER, a new method to identify proteins interacting with DNA sequences in cells. This technique enhances signal detection, aiding in understanding gene regulation and disease-associated DNA variants.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • DNA-protein interactions are crucial for gene regulation.
  • Alterations in these interactions by DNA variants are linked to polygenic diseases.
  • Existing methods for identifying DNA-associated proteins in cells can be slow and have low signal-to-noise ratios.

Purpose of the Study:

  • To develop a faster and more sensitive method for identifying and quantifying proteins associated with specific DNA sequences in living cells.
  • To improve the signal-to-noise ratio (SNR) in proximity proteomics studies.
  • To analyze how DNA variants and mutations affect protein-DNA associations.

Main Methods:

  • Developed Proximal Biotinylation by Episomal Recruitment (PROBER).
  • PROBER utilizes high-copy episomes to amplify the signal.
  • Employs proximity proteomics (BioID) to identify interacting proteins with DNA sequences of interest.

Main Results:

  • PROBER successfully quantified constitutive and inducible associations of transcription factors and chromatin regulators with target DNA sequences.
  • The method identified binding quantitative trait loci influenced by single-nucleotide variants.
  • PROBER revealed altered regulator associations at the hTERT promoter due to cancer hotspot mutations, indicating increased binding of specific gene activators.

Conclusions:

  • PROBER offers a rapid and sensitive approach for identifying proteins bound to specific DNA sequences in living cells.
  • The method can effectively detect changes in protein-DNA interactions caused by genetic variations and mutations.
  • PROBER facilitates the study of gene regulation and the molecular basis of diseases linked to DNA variants.