A boundary-defining protein facilitates megabase-scale regulatory chromosomal loops in Drosophila neurons [Hi-C]

Regulatory elements, such as enhancers and silencers, control transcription by establishing physical proximity to target gene promoters. Neurons in flies and mammals exhibit long-range three-dimensional genome contacts, proposed to connect genes with distal regulatory elements. However, the relevance of these contacts for neuronal gene transcription and the mechanisms underlying their specificity necessitate further investigation. Here, we precisely disrupt several long-range contacts in fly neurons, demonstrating their importance for megabase-range gene regulation and uncovering a hierarchical process in their formation. We further reveal an essential role for the chromosomal boundary-forming protein Cp190 in anchoring many long-range contacts, highlighting a mechanistic interplay between boundary and loop formation. Finally, we develop an unbiased proteomics-based method to systematically identify factors required for specific long-range contacts. Our findings underscore the essential role of architectural proteins such as Cp190 in cell-type-specific genome organization enabling specialized neuronal transcriptional programs. Neurons purified by fluorescence-activated cell sorting (FACS) from old (16-24-hour old) embryos were isolated from mutants or their matched wildtype (WT) controls, and subjected to Hi-C in biological duplicates. More specifically, neurons were immunostained and FACS-purified from CTCF0 or Cp1900 mutants and their matched WT (called WT0) controls. Neurons endogenously expressing mCherry under elav regulatory sequences were FACS-purified from two independent lola-IKO mutants (considered as biological duplicates) and their matched WT controls.