Enhancer identification and activity evaluation in the red flour beetle, Tribolium castaneum

Evolution of cis-regulatory elements (such as enhancers) plays an important role in the production of diverse morphology. However, a mechanistic understanding is often limited by the absence of methods to study enhancers in species outside of established model systems. Here, we sought to establish methods to identify and test enhancer activity in the red flour beetle, Tribolium castaneum. To identify possible enhancer regions, we first obtained genome-wide chromatin profiles from various tissues and stages of Tribolium via FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements)-sequencing. Comparison of these profiles revealed a distinct set of open chromatin regions in each tissue and stage. In addition, comparison of the FAIRE data to sets of computationally predicted (SCRMshaw) enhancers revealed a very high overlap between the two datasets. Second, using nubbin in the wing and hunchback in the embryo as case studies, we established the first universal reporter assay system that works in various contexts in Tribolium, and in a cross-species context. Together, these advances will facilitate investigation of cis-evolution and morphological diversity in Tribolium and other insects.