Chromosomes are folded into highly compacted structures to accommodate physical constraints within nuclei and to regulate access to genomic information. Recently, global mapping of pairwise contacts showed that loops anchoring topological domains (TADs) are highly conserved between cell types and species. Whether pairwise loops synergize to form higher-order structures is still unclear. Here we develop a conformation capture assay to study higher-order organization using chromosomal walks (C-walks) that link multiple genomic loci together into proximity chains in human and mouse cells. This approach captures chromosomal structure at varying scales. Inter-chromosomal contacts constitute only 7-10% of the pairs and are restricted by interfacing TADs. About half of the C-walks stay within one chromosome, and almost half of those are restricted to intra-TAD spaces. C-walks that couple 2-4 TADs indicate stochastic associations between transcriptionally active, early replicating loci. Targeted analysis of thousands of 3-walks anchored at highly expressed genes support pairwise, rather than hub-like, chromosomal topology at active loci. Polycomb-repressed Hox domains are shown by the same approach to enrich for synergistic hubs. Together, the data indicate that chromosomal territories, TADs, and intra-TAD loops are primarily driven by nested, possibly dynamic, pairwise contacts.

Metazoan genomes are partitioned into modular chromosomal domains containing active or repressive chromatin. In flies, Polycomb group (PcG) response elements (PREs) recruit PHO and other DNA-binding factors and act as nucleation sites for the formation of Polycomb repressive domains. The sequence specificity of PREs is not well understood. Here, we use comparative epigenomics and transgenic assays to show that Drosophila domain organization and PRE specification are evolutionarily conserved despite significant cis-element divergence within Polycomb domains, whereas cis-element evolution is strongly correlated with transcription factor binding divergence outside of Polycomb domains. Cooperative interactions of PcG complexes and their recruiting factor PHO stabilize PHO recruitment to low-specificity sequences. Consistently, PHO recruitment to sites within Polycomb domains is stabilized by PRC1. These data suggest that cooperative rather than hierarchical interactions among low-affinity sequences, DNA-binding factors, and the Polycomb machinery are giving rise to specific and strongly conserved 3D structures in Drosophila.