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Cited by 208Genomics and Chromatin DynamicsChromatin Remodeling and CancerCRISPR and Genetic Engineering

Resolving systematic errors in widely used enhancer activity assays in human cells

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Muerdter, Felix, Boryń, Łukasz M, Woodfin, Ashley R, Neumayr, Christoph, Rath, Martina, Zabidi, Muhammad A, Pagani, Michaela, Haberle, Vanja +5 more·2018·Nature Methods
DOI 10.1038/nmeth.4534PMC PMC5793997
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Abstract

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 such assays are rendered unreliable by two previously reported phenomena relating to plasmid transfection into human cells: (i) the bacterial plasmid origin of replication (ORI) functions as a conflicting core promoter and (ii) a type I interferon (IFN-I) response is activated. These cause 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, enabling 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.

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  • The correlation between functional activity measured at scale (e.g. via MPRAs) and the annotations typically used to call enhancers is modest at best.

    “Furthermore, to the extent that functional activity has been measured at scale, e.g. via MPRAs, its correlation with the annotations typically used to call enhancers is modest at best106-108.”

    From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [108]

    The Results section reports that only 42.3% of the 9,613 STARR-seq enhancers were accessible in HeLa-S3 cells, supporting a substantial mismatch with one commonly used annotation. But it also reports significant, 20.5-fold enrichment of STARR-seq peaks in ChromHMM strong-enhancer regions. The paper therefore supports imperfect agreement, not the broader assertion that correlation with enhancer annotations is modest at best. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • Genome-wide 'shotgun MPRAs' avoid a priori assumptions about which sequences to test.

    “In contrast with MPRAs relying on re-synthesis of candidate sequences, genome-wide ‘shotgun MPRAs’108,174,183,188 nicely avoid a priori assumptions about which sequences to test.”

    From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [108]

  • MPRAs have been applied to assess biochemically annotated candidate enhancers, candidate enhancers harbouring variants that potentially mediate eQTLs, and even scans of the entire human genome.

    “MPRAs have been applied to assess biochemically annotated candidate enhancers77,176-178, candidate enhancers harbouring variants that potentially mediate eQTLs179-182, and even scans of the entire human genome108,183.”

    From Towards a comprehensive catalogue of validated and target-linked human enhancers (2020) · cites this paper as [108]

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Research works printed but not linked: 50

  1. [1]Shlyueva D Stampfel G Stark A Transcriptional enhancers: from properties to genome-wide predictions Nat Rev Genet 2014 15 272 286 24614317
  2. [2]Santiago-Algarra D Dao LTM Pradel L España A Spicuglia S Recent advances in high-throughput approaches to dissect enhancer function F1000Res 2017 6 939 28690838 PMC5482341
  3. [3]Lemp NA Hiraoka K Kasahara N Logg CR Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function Nucleic Acids Res 2012 40 7280 7290 22618870 PMC3424574
  4. [4]Zabidi MA Enhancer-core-promoter specificity separates developmental and housekeeping gene regulation Nature 2015 518 556 559 25517091 PMC6795551
  5. [5]Saragosti S Moyne G Yaniv M Absence of nucleosomes in a fraction of SV40 chromatin between the origin of replication and the region coding for the late leader RNA Cell 1980 20 65 73 6248237
  6. [7]Arnold CD Genome-wide assessment of sequence-intrinsic enhancer responsiveness at single-base-pair resolution Nat Biotechnol 2017 35 136 144 28024147 PMC5870828
  7. [8]Juven-Gershon T Cheng S Kadonaga JT Rational design of a super core promoter that enhances gene expression Nat Methods 2006 3 917 922 17124735
  8. [9]Pine R Levy DE Reich N Darnell JE Jr Transcriptional stimulation by CaPO4-DNA precipitates Nucleic Acids Res 1988 16 1371 1378 3126485 PMC336321
  9. [11]Paludan SR Bowie AG Immune sensing of DNA Immunity 2013 38 870 880 23706668 PMC3683625
  10. [12]Bridge AJ Pebernard S Ducraux A Nicoulaz A-L Iggo R Induction of an interferon response by RNAi vectors in mammalian cells Nat Genet 2003 34 263 264 12796781
  11. [13]Dao LTM Genome-wide characterization of mammalian promoters with distal enhancer functions Nat Genet 2017 10.1038/ng.3884 28581502 · DOI · Paper page
  12. [14]Landry JJM The genomic and transcriptomic landscape of a HeLa cell line G3 2013 3 1213 1224 23550136 PMC3737162
  13. [16]Nguyen TA High-throughput functional comparison of promoter and enhancer activities Genome Res 2016 26 1023 1033 27311442 PMC4971761
  14. [17]Chen Q Sun L Chen ZJ Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing Nat Immunol 2016 17 1142 1149 27648547
  15. [18]Chan YK Gack MU Viral evasion of intracellular DNA and RNA sensing Nat Rev Microbiol 2016 14 360 373 27174148 PMC5072394
  16. [19]Nejepinska J Malik R Wagner S Svoboda P Reporters transiently transfected into mammalian cells are highly sensitive to translational repression induced by dsRNA expression PLoS One 2014 9 e87517 24475301 PMC3903663
  17. [20]Nejepinska J Malik R Moravec M Svoboda P Deep sequencing reveals complex spurious transcription from transiently transfected plasmids PLoS One 2012 7 e43283 22916237 PMC3420890
  18. [21]Clark K Plater L Peggie M Cohen P Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IkappaB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation J Biol Chem 2009 284 14136 14146 19307177 PMC2682862
  19. [22]Jammi NV Whitby LR Beal PA Small molecule inhibitors of the RNA-dependent protein kinase Biochem Biophys Res Commun 2003 308 50 57 12890478
  20. [24]Guertin MJ Lis JT Mechanisms by which transcription factors gain access to target sequence elements in chromatin Curr Opin Genet Dev 2013 23 116 123 23266217 PMC3651763
  21. [25]Ernst J Kellis M ChromHMM: automating chromatin-state discovery and characterization Nat Methods 2012 9 215 216 22373907 PMC3577932
  22. [27]White RJ Transcription by RNA polymerase III: more complex than we thought Nat Rev Genet 2011 12 459 463 21540878
  23. [28]Oler AJ Human RNA polymerase III transcriptomes and relationships to Pol II promoter chromatin and enhancer-binding factors Nat Struct Mol Biol 2010 17 620 628 20418882 PMC2945309
  24. [29]Schuettengruber B Chourrout D Vervoort M Leblanc B Cavalli G Genome regulation by polycomb and trithorax proteins Cell 2007 128 735 745 17320510
  25. [30]Bonn S Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development Nat Genet 2012 44 148 156 22231485
  26. [32]Creyghton MP Histone H3K27ac separates active from poised enhancers and predicts developmental state Proc Natl Acad Sci U S A 2010 107 21931 21936 21106759 PMC3003124
  27. [34]Friedli M Trono D The developmental control of transposable elements and the evolution of higher species Annu Rev Cell Dev Biol 2015 31 429 451 26393776
  28. [35]Chuong EB Elde NC Feschotte C Regulatory evolution of innate immunity through co-option of endogenous retroviruses Science 2016 351 1083 1087 26941318 PMC4887275
  29. [36]Kunarso G Transposable elements have rewired the core regulatory network of human embryonic stem cells Nat Genet 2010 42 631 634 20526341
  30. [37]Stamatoyannopoulos JA What does our genome encode? Genome Res 2012 22 1602 1611 22955972 PMC3431477
  31. [38]Li W Notani D Rosenfeld MG Enhancers as non-coding RNA transcription units: recent insights and future perspectives Nat Rev Genet 2016 17 207 223 26948815
  32. [40]Barakat TS Functional dissection of the enhancer repertoire in human embryonic stem cells bioRxiv 2017 146696 10.1101/146696 PMC6084406 30033119 · DOI
  33. [43]Walters AA Comparative analysis of enzymatically produced novel linear DNA constructs with plasmids for use as DNA vaccines Gene Ther 2014 21 645 652 24830436 PMC4082409
  34. [44]Shen SQ Massively parallel cis-regulatory analysis in the mammalian central nervous system Genome Res 2016 26 238 255 26576614 PMC4728376
  35. [45]Inoue F A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity Genome Res 2017 27 38 52 27831498 PMC5204343
  36. [46]Maricque BB Dougherty JD Cohen BA A genome-integrated massively parallel reporter assay reveals DNA sequence determinants of cis-regulatory activity in neural cells Nucleic Acids Res 2017 45 e16 28204611 PMC5389540
  37. [47]Rickels R An Evolutionary Conserved Epigenetic Mark of Polycomb Response Elements Implemented by Trx/MLL/COMPASS Mol Cell 2016 63 318 328 27447986 PMC4996622
  38. [48]Lanoix J Acheson NH A rabbit beta-globin polyadenylation signal directs efficient termination of transcription of polyomavirus DNA EMBO J 1988 7 2515 2522 2847921 PMC457122
  39. [49]Ishida Y Leder P RET: a poly A-trap retrovirus vector for reversible disruption and expression monitoring of genes in living cells Nucleic Acids Res 1999 27 e35 10572187 PMC148758
  40. [50]Vitter JS Random sampling with a reservoir ACM Trans Math Softw 1985 11 37 57
  41. [51]Langmead B Trapnell C Pop M Salzberg SL Ultrafast and memory-efficient alignment of short DNA sequences to the human genome Genome Biol 2009 10 R25 19261174 PMC2690996
  42. [52]Quinlan AR Hall IM BEDTools: a flexible suite of utilities for comparing genomic features Bioinformatics 2010 26 841 842 20110278 PMC2832824
  43. [53]Stark A Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures Nature 2007 450 219 232 17994088 PMC2474711
  44. [56]Bailey TL Gribskov M Combining evidence using p-values: application to sequence homology searches Bioinformatics 1998 14 48 54 9520501
  45. [57]Saldanha AJ Java Treeview--extensible visualization of microarray data Bioinformatics 2004 20 3246 3248 15180930
  46. [58]Harrow J GENCODE: the reference human genome annotation for The ENCODE Project Genome Res 2012 22 1760 1774 22955987 PMC3431492
  47. [61]FitzGerald PC Sturgill D Shyakhtenko A Oliver B Vinson C Comparative genomics of Drosophila and human core promoters Genome Biol 2006 7 R53 16827941 PMC1779564
  48. [62]Ohler U Liao G-C Niemann H Rubin GM Computational analysis of core promoters in the Drosophila genome Genome Biol 2002 3 RESEARCH0087 PMC151189 12537576
  49. [63]Parry TJ The TCT motif, a key component of an RNA polymerase II transcription system for the translational machinery Genes Dev 2010 24 2013 2018 20801935 PMC2939363
  50. [64]Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2-[Delta][Delta] CT method Methods 2001 25 402 408 11846609

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Authors
Muerdter, F., Boryń, Ł. M., Woodfin, A. R., Neumayr, C., Rath, M., Zabidi, M. A., Pagani, M., Haberle, V., Kazmar, T., Catarino, R. R., Schernhuber, K., Arnold, C. D., & Stark, A.
Affiliations
Research Institute of Molecular Pathology (AT)
Corresponding author
Stark, Alexander
ORCID
Venue
Nature Methods
Publisher
Springer Science and Business Media LLC
Published
2017-12-11
Volume
15
Issue
2
Pages
141-149
ISSN
  • 1548-7091 (ISSN-L)
  • 1548-7105
DOI
10.1038/nmeth.4534
PMC
PMC5793997
OpenAlex
W2772841828
Cited by
208
References
12
Citations by year
2026: 20 · 2025: 25 · 2024: 26 · 2023: 21 · 2022: 23 · 2021: 28 · 2020: 32 · 2019: 13 · 2018: 18 · 2017: 2
FWCI
8.05
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature Methods (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Keywords
enhancers, type I interferon response, STARR-seq, HeLa S3 cells, enhancer silencing, luciferase reporter assay
MeSH terms
Chromatin, Chromosome Mapping, Enhancer Elements, Genetic, False Negative Reactions, Gene Expression Regulation, HeLa Cells, Humans, Promoter Regions, Genetic, Genome, Human, Genes, Reporter

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