Single-cell mapping of regulatory DNA-protein interactions

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Gene expression regulation relies on transcription factors (TFs) binding to DNA.
• Mapping TF-DNA interactions, especially weak or transient ones, in single cells is challenging.
• Existing methods struggle to capture the full spectrum of TF binding dynamics.

Purpose of the Study:

• To develop a novel single-cell technology for profiling DNA-protein interactions.
• To overcome limitations in detecting weak or transient TF binding.
• To enable integrated analyses of gene regulation within single cells.

Main Methods:

• Developed docking and deamination followed by sequencing (D&D-seq), a single-cell immuno-tethering technology.
• Coupled an antibody-binding nanobody with a cytosine base editor for targeted DNA editing at protein-bound sites.
• Integrated D&D-seq with ATAC-seq and scATAC-seq for chromatin accessibility and TF activity assessment.

Main Results:

• D&D-seq successfully detects weak or transient TF binding through cytosine-to-uracil editing.
• The technology is compatible with standard single-cell multi-omic workflows.
• Captured CTCF binding across active and inactive chromatin compartments by coupling D&D-seq with whole-genome sequencing.

Conclusions:

• D&D-seq provides a powerful new tool for mapping DNA-protein interactions at single-cell resolution.
• This technology advances the understanding of gene regulation by enabling comprehensive analysis of TF binding dynamics.
• Facilitates integrated studies of epigenetics, chromatin accessibility, and TF activity.