Abstract
The discovery of transcription factors (TFs) controlling pathways in health and disease is of paramount interest. We designed a widely applicable method, dubbed barcorded synthetic tandem repeat promoter screening (BC-STAR-PROM), to identify signal-activated TFs without any a priori knowledge about their properties. The BC-STAR-PROM library consists of ∼3000 luciferase expression vectors, each harboring a promoter (composed of six tandem repeats of synthetic random DNA) and an associated barcode of 20 base pairs (bp) within the 3' untranslated mRNA region. Together, the promoter sequences encompass >400,000 bp of random DNA, a sequence complexity sufficient to capture most TFs. Cells transfected with the library are exposed to a signal, and the mRNAs that it encodes are counted by next-generation sequencing of the barcodes. This allows the simultaneous activity tracking of each of the ∼3000 synthetic promoters in a single experiment. Here we establish proof of concept for BC-STAR-PROM by applying it to the identification of TFs induced by drugs affecting actin and tubulin cytoskeleton dynamics. BC-STAR-PROM revealed that serum response factor (SRF) is the only immediate early TF induced by both actin polymerization and microtubule depolymerization. Such changes in cytoskeleton dynamics are known to occur during the cell division cycle, and real-time bioluminescence microscopy indeed revealed cell-autonomous SRF-myocardin-related TF (MRTF) activity bouts in proliferating cells.
Highlights
The identification of transcription factors (TFs) responding to a specific signal is one of the first steps in dissecting the underlying regulatory networks
Two sequencing strategies were used to associate the barcodes with the corresponding promoter repeats: SMRT sequencing (Pacific Biosciences), which, owing to high read lengths, allowed the complete sequencing of a 2.3-kb restriction fragment encompassing both the barcode and the promoter repeats, and Illumina sequencing of restriction fragments, in which barcodes and promoter repeats were brought into proximity by intramolecular ligation (Material and Methods; Supplemental Fig. S2A)
We showed that BC-STAR-PROM allows the identification of cis-acting elements for TFs responding to any stimulus of interest
Summary
The identification of transcription factors (TFs) responding to a specific signal is one of the first steps in dissecting the underlying regulatory networks. A successful and unbiased experimental strategy was developed by Gerber et al (2013) to screen for TFs induced by blood-borne signals This method, dubbed synthetic tandem repeat promoter screening (STAR-PROM) involves the construction of a luciferase reporter gene library with tandemly repeated synthetic promoter elements. It relies on the observation that most TF-binding sites exist at a relatively high frequency in random DNA (Reinke et al 2008; Gerber et al 2013) This strategy requires thousands of transfections of individual expression vectors and is extremely time-consuming and labor-intensive. As suggested by single-cell bioluminescence recordings, such changes in cytoskeleton dynamics during the cell division cycle could elicit cell-autonomous bursts of SRF–MRTF activation
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