Decoding chromosome organization using proximity labeling and long-read sequencing

Area of Science:

• Genomics
• Molecular Biology
• Chromatin Biology

Background:

• Current genomic methods often fail to preserve crucial connectivity information, hindering the characterization of large-scale genome organization.
• Understanding DNA-protein interactions and chromosome architecture is vital for deciphering cellular processes.

Purpose of the Study:

• To develop a novel in vivo proximity-labeling technique, npDamID, for indelibly marking and decoding protein-associated DNA sites.
• To characterize large-scale genome organization and protein association patterns, particularly in challenging repetitive regions.

Main Methods:

• npDamID utilizes a tethered dam methyltransferase to label DNA in proximity to a protein of interest.
• Nanopore sequencing of ultra-long reads (>100 kb) identifies methylated bases, enabling the reconstruction of DNA-protein interaction landscapes.
• The technique was validated in budding yeast by analyzing the cohesin-based meiotic backbone.

Main Results:

• npDamID successfully recapitulated known cohesin association patterns and revealed cell-to-cell variability.
• Analysis of single reads demonstrated distance-dependent correlations between methylated sites.
• The method accurately mapped in vivo cohesin association within the repetitive ribosomal DNA locus by anchoring on unique regions.

Conclusions:

• npDamID is a versatile technique for mapping heterogeneous protein association patterns and in vivo chromosome conformations.
• This approach overcomes limitations of existing methods, particularly in repetitive genomic regions.
• npDamID promises to advance our understanding of diverse chromosomal processes and genome organization.