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

Abstract

Evolution of -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, To identify possible enhancer regions, we first obtained genome-wide chromatin profiles from various tissues and stages of 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-regulatory module-predicted) enhancers revealed a very high overlap between the two datasets. Second, using in the wing and in the embryo as case studies, we established the first universal reporter assay system that works in various contexts in , and in a cross-species context. Together, these advances will facilitate investigation of-evolution and morphological diversity in and other insects.

How to study enhancers in non-traditional insect models

“…When SCRMshaw-predicted enhancers from other insects, including bees, wasps, beetles and mosquitoes, are tested in reporter gene assays in transgenic Drosophila, they validate at rates similar to those seen from within-species prediction of Drosophila enhancers (Kazemian et al, 2014;Suryamohan et al, 2016). Direct testing of Tribolium enhancers in transgenic Tribolium confirms that SCRMshaw can find bona fide enhancers cross-species (Lai et al, 2018). Although not tested in insects, Chen et al (2018) similarly demonstrate that a k-mer-based prediction method trained using data from a single species can be used for enhancer discovery across a range of mammalian genomes.…”

“…2D) red flour beetle (Tribolium castaneum) and successfully obtained genome-wide chromatin profiles from various tissues and stages of this insect (Fig. 4) (Lai et al, 2018). More than 40,000 open chromatin regions in the Tribolium genome are detected, and comparison of the profiles across the samples revealed a distinct set of open chromatin regions in each tissue and at each stage.…”

“…In Tribolium, we compared the FAIRE profiles with SCRMshaw predictions and found surprisingly high overlaps between these two datasets (Fig. 4) (Lai et al, 2018). However, in the case of Tribolium (but we think this can be generalizable), chromatin profiling provided too many candidate regions (>40,000 Box 1.…”

“…For example, enhancers of some developmental genes in beetles, honeybees and even spiders were demonstrated to be active in their expected contexts in Drosophila (e.g. Ayyar et al, 2010;Cande et al, 2009a,b;Kazemian et al, 2014;Lai et al, 2018;Prasad et al, 2016;Wolff et al, 1998;Zinzen et al, 2006). These studies show the power of the cross-species reporter assay using Drosophila as an in vivo test tube, but with one unavoidable concern: are these non-Drosophila enhancers really showing biologically relevant activities in Drosophila?…”

“…Based on the extensive characterization of core promoter compatibility in Tribolium by Schinko et al (2010), we thought that the Tc-bhsp68 core promoter would be a safe choice when we attempted to establish a reporter assay system in Tribolium. However, surprisingly, the Tc-bhsp68 core promoter failed to work reliably in Tribolium in a reporter construct with a wing enhancer of the nubbin gene (Tc-nub), even though the same construct worked well in Drosophila (Lai et al, 2018). We decided to try DSCP (Drosophila Synthetic Core Promoter), the core promoter that was established for a genome-wide reporter assay in Drosophila (the FlyLight project) (Pfeiffer et al, 2008).…”