Linking Cellular Signalling to Gene Expression Using EXT-Encoded Reporter Libraries

Intracellular signalling initiated by extracellular ligands that activate cell surface receptors is a complicated process that involves multiple interconnected biochemical steps. Protein–protein interactions are often regulated by activated kinases via phosphorylation of specific residues. Such transient regulated interactions are central to many signalling cascades. Downstream signalling converges at the level of transcription factors to finally regulate adaptive transcriptional responses. There are powerful methods available to study transcriptional changes even at a global level; however, measuring upstream regulatory mechanisms is still challenging. We designed an experimental approach termed ex pressed oligonucleotide t ag (EXT)assay that enables the parallel analysis of signalling events upstream of gene expression. We make use of different types of reporter gene assays that are invariably linked to unique EXTs serving as quantitative decoders of respective assays. EXT reporters can be introduced into living cells and analyzed in pools by microarray hybridization or sequencing.