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

ABSTRACT Evolution of cis -properties (such as enhancers) often 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. Second, we established the first reporter assay system that works in both Drosophila and Tribolium , using nubbin in the wing and hunchback in the embryo as case studies. Together, these advances will be useful to study the evolution of cis -language and morphological diversity in Tribolium and other insects.