Mapping single-cell diploid chromatin fiber architectures using DAF-seq.

Area of Science:

• Genomics
• Epigenetics
• Molecular Biology

Background:

• Gene regulation relies on protein co-binding on chromatin fibers within cells.
• Understanding protein occupancy heterogeneity across haplotypes and cells in diploid organisms is challenging.

Purpose of the Study:

• To develop a novel method for high-resolution, single-molecule analysis of protein occupancy on chromatin.
• To investigate chromatin plasticity and protein co-regulation at the single-cell and single-haplotype level.

Main Methods:

• Deaminase-Assisted single-molecule chromatin Fiber sequencing (DAF-seq) was developed.
• DAF-seq provides near-nucleotide resolution for single-molecule footprinting, chromatin states, and DNA sequence profiling.
• Single-cell DAF-seq (scDAF-seq) was applied to generate genome-wide co-occupancy maps.

Main Results:

• DAF-seq enables illumination of cooperative protein occupancy at regulatory elements.
• scDAF-seq maps protein co-occupancy across 99% of the mappable genome in single cells.
• Significant chromatin plasticity was observed, with 61% divergence between haplotypes and 63% between cells.
• Regulatory elements show distance-dependent co-actuation, mirroring cohesin-mediated loops.

Conclusions:

• DAF-seq provides unprecedented single-nucleotide, single-molecule, single-haplotype, and single-cell precision for characterizing protein occupancy.
• The study reveals extensive chromatin plasticity and provides insights into the functional impact of somatic variants and epialleles.
• Findings highlight the dynamic nature of gene regulation at the molecular level in diploid genomes.