Capturing pairwise and multi-way chromosomal conformations using chromosomal walks.

Area of Science:

• Genomics
• Molecular Biology
• Cell Biology

Background:

• Chromosomes fold into compact structures for nuclear space and gene regulation.
• Pairwise contacts form loops anchoring topological domains (TADs), conserved across species.
• The formation of higher-order structures from these loops remains unclear.

Purpose of the Study:

• To investigate higher-order chromosomal organization beyond pairwise contacts.
• To develop and apply a novel conformation capture assay (chromosomal walks) for studying genome structure at multiple scales.
• To determine whether pairwise loops synergize into higher-order structures.

Main Methods:

• Development of chromosomal walks (C-walks), a conformation capture assay linking multiple genomic loci.
• Application of C-walks in human and mouse cells to capture chromosomal structure at varying scales.
• Analysis of inter-chromosomal contacts, intra-chromosomal contacts, and intra-TAD interactions.

Main Results:

• Inter-chromosomal contacts are limited (7-10%) and restricted by TADs.
• Approximately half of C-walks occur within a single chromosome, with many confined to intra-TAD spaces.
• C-walks coupling 2-4 TADs suggest stochastic associations in transcriptionally active, early replicating loci.
• Highly expressed genes show pairwise topology, while Polycomb-repressed Hox domains exhibit synergistic hubs.

Conclusions:

• Chromosomal territories, TADs, and intra-TAD loops are primarily formed by nested, potentially dynamic, pairwise contacts.
• Active and repressed genomic regions display distinct higher-order organizational principles.
• The study provides insights into the hierarchical folding of the genome and its regulation.