Genome-wide mapping of autonomous promoter activity in human cells

Abstract

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 to assay more than 108 DNA fragments, each 0.2–2kb in size, for their ability to drive transcription autonomously. In SuRE, a plasmid library is constructed of random genomic fragments upstream of a 20bp barcode and decoded by paired-end sequencing. This library is then transfected into cells and transcribed barcodes are quantified in the RNA by high throughput sequencing. When applied to the human genome, we achieved a 55-fold genome coverage, allowing us to map autonomous promoter activity genome-wide. By computational modeling we delineated subregions within promoters that are relevant for their activity. For instance, 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.

The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription

“…A number of studies have measured the ability of different genomic elements to function as either enhancers or promoters using massively parallel cell culture-based reporter assays. Measuring promoter activity of thousands of small DNA elements (Nguyen et al 2016) or across the entire human genome (van Arensbergen et al 2017) suggests that many enhancers (as well as repetitive elements) can act as weak autonomous promoters, although at levels ∼10-fold lower than that of annotated gene promoters. Conversely, testing promoters for enhancer activity revealed that ∼3% of human elements (Dao et al 2017) and 4.5% of Drosophila elements (Arnold et al 2013) spanning promoters have in vitro enhancer activity.…”

Core promoters across the genome

“…This is now possible using either of the new methods---self-transcribing active core promoter-sequencing (STAP-seq) by Arnold et al 7 ( Fig. 1a) and survey of regulatory elements (SuRE) by van Arensbergen et al 8 (Fig. 1b).…”

“…The authors construct a candidate libraryand use it to assess sequence-intrinsic core promoter strength and promoter potential of candidate regions that span the entire Drosophila genome (Figure 1a In the second paper, van Arensbergen et al 8 introduce SuRE, which similarly queries the autonomous promoter activity of random genomic fragments outside of their genomic context using a defined reporter assay. One important difference relative to STAP-seq is the size of the tested fragments: to allow the entire human genome to be probed at high coverage, A limitation of SuRE compared to STAP-seq is lower resolution: the peaks obtained correspond in size to the tested fragments, whereas STAP-seq maps initiation start sites at single-nucleotide resolution.…”

“…One important difference relative to STAP-seq is the size of the tested fragments: to allow the entire human genome to be probed at high coverage, A limitation of SuRE compared to STAP-seq is lower resolution: the peaks obtained correspond in size to the tested fragments, whereas STAP-seq maps initiation start sites at single-nucleotide resolution. However, because the tested fragments overlap, van Arensbergen et al 8 are able to computationally enhance the resolution of SuRE signals by determining the minimal regions essential for reporter expression.…”

“…van Arensbergen et al 8 focus on exploring promoter autonomy, which they define as the ratio of autonomous to endogenous transcription initiation (i.e., the ratio of SuRE to GRO-cap 5 signals). Although the trends are noisy, promoter autonomy is overall greater in ubiquitously expressed genes that have a lower dependence on the number of endogenous enhancers attributed to them.…”