<i>Cis</i>-regulatory modes of <i>Ultrabithorax</i> inactivation in butterfly forewings.

Abstract

<i>Hox</i> gene clusters encode transcription factors that drive regional specialization during animal development: for example the Hox factor Ubx is expressed in the insect metathoracic (T3) wing appendages and differentiates them from T2 mesothoracic identities. <i>Hox</i> transcriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other than <i>Drosophila</i>, which shows a derived <i>Hox</i> dislocation into two genomic clusters that disjoined <i>Antennapedia</i> (<i>Antp</i>) and <i>Ultrabithorax</i> (<i>Ubx</i>). Here, we investigated how <i>Ubx</i> is restricted to the hindwing in butterflies, amidst a contiguous <i>Hox</i> cluster. By analysing Hi-C and ATAC-seq data in the butterfly <i>Junonia coenia</i>, we show that a Topologically Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin opening around <i>Ubx</i>. This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around the <i>Antp</i> locus. CRISPR mutational perturbation of this BE releases ectopic <i>Ubx</i> expression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putative <i>cis-</i>regulatory module within the <i>Ubx</i> TAD 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 in <i>Heliconius</i> butterflies, and discuss their possible mutational basis. By leveraging the extensive wing specialization found in butterflies, our initial exploration of <i>Ubx</i> regulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings.