STARR-Seq

Self-transcribing active regulatory region sequencing (STARR-seq) is a method of directly discovering and quantitively assessing enhancer activity on a genome-wide scale. STARR-seq identifies active, chromatin-masked, and dormant enhancers by assaying enhancer activity of genomic fragments episomally. Briefly, genomic DNA is fragmented, and linkers are added to fragment ends. This library of fragments is then cloned into a vector downstream of a core promoter, the vector library is transfected into cells, and after 24 h, RNA is harvested, and a cDNA library generated. Genomic DNA is simultaneously harvested to control for differential transfection efficiencies. Cloned fragments with enhancer activity will drive expression of themselves and resulting sequence output will both identify enhancers and quantify their activity. This method allows for the simultaneous screening of the entire genome for enhancer activity [95,102,106]. There are a number of available methods for analysis of STARR-seq data and identification of enhancer peaks [107,108]. Drawbacks of STARR-seq are twofold; the first being that many enhancers are “context dependent”, meaning that their position in the genome is important, and the STARR-seq approach removes DNA fragments from their genomic context. Enhancers may interact with other nearby regulatory elements, or distal regulatory elements that are brought to interact with an enhancer through changes in the chromatin structure. The second being that this method discovers all enhancers within the tested DNA, making it difficult to determine which enhancers are relevant to a condition [105]. Despite these limitations, this method, capitalizing on next-generation sequencing approaches to comprehensively query enhancer activity on a genome-wide scale, generates a comprehensive catalog of an organism’s enhancers.

STARR-seq has been used in Drosophila to comprehensively characterize and compare transcriptional enhancers across five closely-related species [95]. This seminal work concludes that there is a good degree of evolutionary conservation in enhancer activity, as well as frequent gains in enhancer function since divergence from the common ancestor. Work in An. coluzzii has examined the impact of naturally-segregating genetic variation in a small number of enhancers with potential roles in mosquito development, immunity, and insecticide resistance [109].