Multiscale spatial analysis implicates chromosomal metaloops in gene patterning across the Drosophila brain

Scores of chromosome-scale loops, or metaloops, arise in the Drosophila brain, but their spatial organization and relationship to neural gene expression patterns remain unclear. Here, we used multiplexed Optical Reconstruction of Chromatin Architecture (ORCA) to examine the multiscale spatial organization of metaloops in cross-sections of 100s of larval and adult Drosophila brains. We find metaloops form preferentially in the central regions of the brain, where they nucleate the formation of metadomains, characterized by the intermingling of distal topologically associating domains (TADs). At the sub-cellular scale, metaloops tend to arise towards the nuclear center, and multiple metaloops in the same cell have a preference to form hubs (3 or more contacts). Each brain nucleus generally harbors only a few loops or hubs. An in-depth analysis of the hub centered on DIP-epsilon, a synaptic wiring gene, identified a three-way metadomain that brings together the DIP-epsilon TAD; a distal TAD carrying a paralog of DIP-epsilon, DIP-zeta; and a putative regulatory TAD, across 3 Mb. This metadomain adopts distinct conformations depending on gene expression; cells expressing DIP-epsilon or DIP-zeta show preferential interactions between the TAD carrying the corresponding gene and the putative regulatory TAD. We posit that the neuron-specific formation of different subsets of metadomains might coordinate the expression of diverse combinations of synaptic wiring genes underlying complex brain architecture.