Author:

Abstract:

Deciphering the cis-regulatory logic encoded in enhancer sequences requires large-scale reporter assays to experimentally validate candidate enhancers predicted by genomic approaches such as chromatin accessibility and ChIP-seq. Here, we propose a novel high-throughput enhancer-reporter assay called CHEQ-Seq (Captured High-throughput Enhancer testing by Quantitative Sequencing). A set of candidate enhancers are pre-selected as regions of 0.5-1 kb and enriched from genomic, sheared DNA using custom-designed capturing baits. They are subsequently cloned into a reporter library and randomly combined with unique barcodes, before being tested under various conditions in cell culture. The relationship between each enhancer and its reporter-barcode is determined by PacBio long-read sequencing of the entire library; while the barcode expression level is determined by Illumina short-read cDNA sequencing. We have applied Cheq-seq to test the enhancer activity of 1526 p53 ChIP-seq peaks under p53 knock-down and p53 over-activating conditions. We obtained reproducible reporter expression for 1060 captured enhancers, of which 397 showed a significant p53-dependent activation. Strikingly, the large majority (99%) of p53 target enhancers can be characterized and distinguished from negative sequences by the occurrence of a single p53 binding site. Thus, the p53 enhancer logic represents a new ancestral class of enhancers, distinct from developmental enhancers that adhere to the billboard and enhanceosome models. The p53 enhancers do not contain obvious combinatorial complexity of binding sites for multiple transcription factors. This suggests that p53 acts alone on its target enhancers, and that context-dependent regulation of target genes is not encoded in the p53 enhancer sequences, but at different upstream or downstream layers of the cell’s gene regulatory network.