Marion Mouginot1, Sahar Hani1, Pascal Cousin1, Julien Dorier2,3, Arianna Ravera4 and Maria Cristina Gambetta1 1Center for Integrative Genomics, University of Lausanne, 1015 Lausanne, Switzerland; 2Bioinformatics Competence Center, University of Lausanne, 1015 Lausanne, Switzerland; 3Bioinformatics Competence Center, Swiss Federal Institute of Technology Lausanne, 1015 Lausanne, Switzerland; 4Scientific Computing and Research Support Unit, University of Lausanne, 1015 Lausanne, Switzerland Corresponding author: mariacristina.gambettaunil.ch Abstract

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 in enabling specialized neuronal transcriptional programs.

Received January 19, 2025. Accepted March 19, 2025.