Enhancer identification and activity evaluation in the red flour beetle, <i>Tribolium castaneum</i>

Abstract

Evolution of <i>cis</i>-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 for studying enhancers in species other than established model systems. Here, we sought to establish methods to identify and test enhancer activity in the red flour beetle, <i>Tribolium castaneum</i> To identify possible enhancer regions, we first obtained genome-wide chromatin profiles from various tissues and stages of <i>Tribolium</i> using FAIRE (formaldehyde-assisted isolation of regulatory elements)-sequencing. Comparison of these profiles revealed a distinct set of open chromatin regions in each tissue and at each stage. In addition, comparison of the FAIRE data with sets of computationally predicted (i.e. supervised <i>cis</i>-regulatory module-predicted) enhancers revealed a very high overlap between the two datasets. Second, using <i>nubbin</i> in the wing and <i>hunchback</i> in the embryo as case studies, we established the first universal reporter assay system that works in various contexts in <i>Tribolium</i>, and in a cross-species context. Together, these advances will facilitate investigation of <i>cis</i>-evolution and morphological diversity in <i>Tribolium</i> and other insects.