Cohesin forms fountains at active enhancers in .

Transcriptional enhancers must locate their target genes with both precision and efficiency. In mammals, this specificity is facilitated by topologically associated domains (TADs), which restrict the enhancer search space through three-dimensional genome organization. In contrast, the nematode genome lacks such TAD-based segmentation despite harboring over 30'000 sequences with chromatin signature characteristic of enhancers, thereby raising the question of how enhancer-promoter specificity is achieved. Using high-resolution Hi-C in , we identify distinct 3D chromatin structures surrounding active enhancers, which we term fountains. These structures span 38 kb in average, are unique to active enhancers, and are enriched for the major somatic cohesin complex. Fountains collapse upon cohesin cleavage, indicating their cohesin dependency. Notably, fountains accumulate topological stress, as evidenced by the enrichment of topoisomerases and the psoralen-binding signature of negatively-supercoiled DNA. Functionally, fountain disassembly correlates with transcriptional upregulation of active enhancer-proximal genes, suggesting that fountains act as spatial repressors of enhancer activity. This repression is particularly pronounced for neuronal genes, including the gene, which becomes upregulated, switches isoform and transcription start site upon cohesin loss in a pair of head neurons. Behaviorally, cohesin cleavage alters nematode movement and foraging behavior, linking enhancer-driven transcriptional changes to neural circuit function and organismal phenotypes, reminiscent of pathologies caused by cohesin mutations in humans. Together, our findings uncover fountains as a novel 3D chromatin feature that modulates enhancer activity in a TAD-less genome, establishing a mechanistic link between genome architecture, gene regulation and behavior.