Joris van Arensbergen, Vincent D. FitzPatrick, Marcel de Haas, Ludo Pagie, Jasper Sluimer et al.
Previous methods to systematically characterize sequence-intrinsic activity of promoters have been limited by relatively low throughput and the length of sequences that could be tested. Here we present 'survey of regulatory elements' (SuRE), a method that assays more than 10^8 DNA fragments, each 0.2-2 kb in size, for their ability to drive transcription autonomously. In SuRE, a plasmid library of random genomic fragments upstream of a 20-bp barcode is constructed, and decoded by paired-end sequencing. This library is used to transfect cells, and barcodes in transcribed RNA are quantified by high-throughput sequencing. When applied to the human genome, we achieve 55-fold genome coverage, allowing us to map autonomous promoter activity genome-wide in K562 cells. By computational modeling we delineate subregions within promoters that are relevant for their activity. We show that antisense promoter transcription is generally dependent on the sense core promoter sequences, and that most enhancers and several families of repetitive elements act as autonomous transcription initiation sites.
Joris van Arensbergen, Vincent D. FitzPatrick, Marcel de Haas, Ludo Pagie, Jasper Sluimer, Harmen J. Bussemaker, Bas van Steensel. Genome-wide mapping of autonomous promoter activity in human cells. 2016-12-26. https://doi.org/10.1038/nbt.3754
A genome-wide library of randomly sheared human genomic fragments (approximately 0.2-2 kb) was cloned upstream of a promoter-less reporter transcription unit containing a random 20-bp barcode and transiently transfected into K562 erythroleukemia cells. The processed table contains the authors' called SuRE autonomous-promoter peaks and their genomic feature annotations.
A focused SuRE library made from nine human BACs spanning approximately 1.3 Mb was cloned as random 0.1-1 kb genomic fragments and transiently transfected into K562 cells. Duplicate released coordinate/strand records were merged, then plasmid-input PCR replicates were combined with basal K562, solvent-control, and 50 uM hemin RNA barcode counts to report normalized fragment activity and the hemin response.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
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