Cis-regulatory modes ofUltrabithoraxinactivation in butterfly forewings

Hoxgene clusters encode transcription factors that drive regional specialization during animal development:e.g.the Hox factor Ubx is expressed in the insect metathoracic (T3) wing appendages and differentiates them from T2 mesothoracic identities.Hoxtranscriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other thanDrosophila, which shows a derivedHoxdislocation into two genomic clusters that disjoinedAntennapedia(Antp) andUltrabithorax(Ubx). Here we investigated howUbxis restricted to the hindwing in butterflies, amidst a contiguousHoxcluster. By analysing Hi-C and ATAC-seq data in the butterflyJunonia coenia, we show that a Topologically Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin opening aroundUbx. This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around theAntplocus. CRISPR mutational perturbation of this BE releases ectopicUbxexpression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putativecis-regulatory module within theUbxTAD cause rare homeotic transformations in both directions, indicating the presence of both activating and repressing chromatin features. We also describe a series of spontaneous forewing homeotic phenotypes obtained inHeliconiusbutterflies, and discuss their possible mutational basis. By leveraging the extensive wing specialization found in butterflies, our initial exploration ofUbxregulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings.