Single-cell mapping of regulatory DNA:Protein interactions.

Area of Science:

• Epigenetics and Genomics
• Molecular Biology
• Single-cell Analysis

Background:

• Gene expression is regulated by transcription factors (TFs) and epigenetic signals.
• Disruptions in these networks are linked to aging, disease, and cancer.
• Existing methods limit profiling of DNA:protein interactions in single cells, hindering understanding of regulatory networks.

Purpose of the Study:

• To develop a novel single-cell technology for mapping DNA:protein interactions.
• To overcome limitations of existing methods for profiling transcription factor binding in single cells.
• To enable multimodal analysis of gene regulation in health and disease contexts.

Main Methods:

• Developed Docking & Deamination followed by sequencing (D&D-seq), a single-cell immuno-tethering DNA:protein mapping technology.
• Coupled antibody-binding nanobodies with cytosine base editors to profile TF binding.
• Integrated D&D-seq into single-cell multiomics workflows.

Main Results:

• D&D-seq accurately profiled transcription factor binding (CTCF, GATA) in bulk and single cells with high specificity and efficiency.
• The technique showed minimal off-target activity and high concordance with bulk ChIP-seq.
• Applied to primary human cells, D&D-seq identified CTCF binding sites, predicted 3D chromatin structure, and revealed altered CTCF binding in cells with IDH2 mutations.

Conclusions:

• D&D-seq is a transformative technology for direct mapping of TF and chromatin remodeler binding to DNA in primary human samples.
• This advance opens new avenues for studying chromatin and transcriptional regulation in health and disease.
• Enables deeper understanding of regulatory network perturbations in pathological contexts.