Characterisation of cis-regulatory elements and genome wide enhancer prediction in Heliconiini butterflies

The large-scale annotation and spatiotemporal characterisation of cis-regulatory promoters and enhancers remain limited in many non-model organisms, largely due to fragmented availability of multiomic datasets across species and developmental conditions. Heliconius butterflies, which have been used to investigate wing development and patterning, have provided key insights into the genetic architecture underlying morphological diversification and adaptive pigmentation. Despite this, we are limited in what we understand of how cis-regulatory dynamics drive phenotypic wing patterning variation. Here, by re-analysing and re-evaluating available data, we present a comprehensive new description of a cis-regulatory atlas for Heliconius wing development by integrating chromatin accessibility dynamics (ATAC-seq) with histone modification profiling (H3K27ac, H3K4me3). High-confidence peak sets enabled robust annotation of promoters and enhancers, revealing regulatory distributions consistent with canonical regulatory organisation. Promoters displayed comparatively higher conservation of transcription factor motifs, whilst enhancers exhibited greater sequence diversity, broader genomic distributions and more variable motif repertoires. Comparative analysis revealed cis-regulatory divergence to be primarily temporal, with substantial chromatin remodelling and differential histone enrichment across development, while regulatory dynamics were highly conserved between forewing and hindwing tissues. Extending these experimentally characterised regulatory landscapes, a convolutional neural network was trained for sequence-based enhancer prediction, achieving test AUROC and AUPRC values of 0.909 and 0.913, respectively. Predictions across 34 Heliconiini assemblies displayed significant enrichment within independently characterised accessible chromatin and conserved non-exonic elements (CNEEs), supporting their putative regulatory function. Overall, this study provides a large-scale characterisation of cis-regulatory dynamics and extends enhancer annotation across Heliconiini, providing a foundation for comparative investigations of regulatory conservation and diversification.