Annelien Verfaillie, Dmitry Svetlichnyy, Hana Imrichova, Kristofer Davie, Mark Fiers et al.
Transcription factors regulate their target genes by binding to regulatory regions in the genome. Although the binding preferences of TP53 are known, it remains unclear what distinguishes functional enhancers from nonfunctional binding. In addition, the genome is scattered with recognition sequences that remain unoccupied. Using two complementary techniques of multiplex enhancer-reporter assays, we discovered that functional enhancers could be discriminated from nonfunctional binding events by the occurrence of a single TP53 canonical motif. By combining machine learning with a meta-analysis of TP53 ChIP-seq data sets, we identified a core set of more than 1000 responsive enhancers in the human genome. This TP53 cistrome is invariably used between cell types and experimental conditions, whereas differences among experiments can be attributed to indirect nonfunctional binding events. Our data suggest that TP53 enhancers represent a class of unsophisticated cell-autonomous enhancers containing a single TP53 binding site, distinct from complex developmental enhancers that integrate signals from multiple transcription factors.
Annelien Verfaillie, Dmitry Svetlichnyy, Hana Imrichova, Kristofer Davie, Mark Fiers, Zeynep Kalender Atak, Gert Hulselmans, Valerie Christiaens, Stein Aerts. Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic. 2016-05-18. https://doi.org/10.1101/gr.204149.116
Captured genomic fragments centered on TP53 ChIP-seq peaks, together with housekeeping and negative controls, were cloned upstream of a Venus reporter with 17-bp random barcodes and assayed in MCF7 cells under p53-high (wild-type TP53 plus Nutlin-3a) and p53-low (stable TP53 knockdown) conditions. The table contains fragment-level aggregates of uniquely mapped barcodes and matched plasmid/input and cDNA counts.
The targeted genomic fragments used for CHEQ-seq were inserted downstream of the transcription start site in a self-transcribing STARR-seq reporter and assayed in MCF7 cells under p53-high (wild-type TP53 plus Nutlin-3a) and p53-low (stable TP53 knockdown) conditions. The table contains fixed-length genomic fragments with matched plasmid and reporter cDNA counts and re-derived activity effects.
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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