Felix Muerdter, Łukasz M Boryń, Ashley R Woodfin, Christoph Neumayr, Martina Rath et al.
The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that two previous observations relating to plasmid-transfection into human cells render such assays unreliable: (1) the function of the bacterial plasmid origin-of-replication (ORI) as conflicting core-promoter and (2) the activation of a type-I-interferon (IFN-I) response. These problems cause strongly confounding false-positives and -negatives in luciferase assays and STARR-seq screens. We overcome both problems by employing the ORI as core-promoter and by inhibiting two IFN-I-inducing kinases. This corrects luciferase assays and enables genome-wide STARR-seq screens in human cells. In HeLa-S3 cells, we uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells, and are key to the characterization of human enhancers.
Felix Muerdter, Łukasz M Boryń, Ashley R Woodfin, Christoph Neumayr, Martina Rath, Muhammad A Zabidi, Michaela Pagani, Vanja Haberle, Tomáš Kazmar, Rui R Catarino, Katharina Schernhuber, Cosmas D Arnold, Alexander Stark. Resolving systematic errors in widely-used enhancer activity assays in human cells enables genome-wide functional enhancer characterization. 2017-12-11. https://doi.org/10.1038/nmeth.4534
The same 1–1.5 kb size-selected human genomic DNA library was cloned into the ORI-based human STARR-seq vector and electroporated into HeLa-S3 cells without kinase inhibitors. The processed table contains the published genome-wide peak calls from the untreated comparison screen.
This derived comparison tests differential activity between the inhibitor-treated and untreated HeLa-S3 ORI STARR-seq screens described above. Peak calls overlapping by at least 85% were collapsed and evaluated with the paper's hypergeometric differential test; the table preserves all published comparison intervals and labels the FDR-supported direction.
A genome-wide library of 1–1.5 kb size-selected human genomic DNA fragments was cloned into the ORI-based human STARR-seq vector and electroporated into HeLa-S3 cells. Cells received 1 µM PKR inhibitor C16 and 1 µM TBK1/IKK inhibitor BX-795 immediately after electroporation; the processed table contains the published peak calls from the inhibitor screen.
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.
Download all 5 files (ZIP)GSE100432_differential_peaks_inhibitor_vs_no-inhibitor_supp.table2.tsv.gzGSE100432_family.soft.gzGSE100432_peaks_inhibitor_correctedEnrichment4_supp.table2.tsv.gzGSE100432_peaks_no-inhibitor_correctedEnrichment4_supp.table2.tsv.gzGSE100432_shortlisted_regions_inhibitor_supp.table2.tsv.gz