Determinants of enhancer and promoter activities of regulatory elements

Abstract

The proper activities of enhancers and gene promoters are essential for coordinated transcription within a cell. Although diverse methodologies have been developed to identify enhancers and promoters, most have tacitly assumed that these elements are distinct. However, studies have unexpectedly shown that regulatory elements may have both enhancer and promoter functions. Here we review these results, focusing on the factors that determine the promoter and/or enhancer activity of regulatory elements. We discuss emerging models that define regulatory elements by accessible DNA and their non-mutually-exclusive abilities to drive transcription initiation (promoter activity) and/or to enhance transcription at other such regions (enhancer activity).

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Fig. 1: Features used to distinguish promoters and enhancers.
Fig. 2: Chromatin features used to identify and distinguish enhancers and promoters are correlated with the promoter activity of regions, regardless of genomic location.
Fig. 3: General model of regulatory elements and of features associated with promoter and enhancer potential.
Fig. 4: A TF and RNAPII-centric cooperative model of transcriptional regulation.

References

  1. 1.

    Maurano, M. T. et al. Systematic localization of common disease-associated variation in regulatory DNA. Science 337, 1190–1195 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Roadmap Epigenomics Consortium et al. Integrative analysis of 111 reference human epigenomes. Nature 518, 317–330 (2015).

    PubMed Central  Google Scholar 

  3. 3.

    Andersson, R. et al. An atlas of active enhancers across human cell types and tissues. Nature 507, 455–461 (2014). This is the largest study to date defining active enhancers from divergent TSSs of eRNAs. It demonstrates exosome-mediated decay of eRNAs and describes transcribed enhancers as being better validated in in vitro reporter assays than enhancers predicted from histone modifications.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Chatterjee, S. & Ahituv, N. Gene regulatory elements, major drivers of human disease. Annu. Rev. Genomics Hum. Genet. 18, 45–63 (2017).

    CAS  PubMed  Google Scholar 

  5. 5.

    Bradner, J. E., Hnisz, D. & Young, R. A. Transcriptional addiction in cancer. Cell 168, 629–643 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Miguel-Escalada, I., Pasquali, L. & Ferrer, J. Transcriptional enhancers: functional insights and role in human disease. Curr. Opin. Genet. Dev. 33, 71–76 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Haberle, V. & Stark, A. Eukaryotic core promoters and the functional basis of transcription initiation. Nat. Rev. Mol. Cell Biol. 19, 621–637 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Shlyueva, D., Stampfel, G. & Stark, A. Transcriptional enhancers: from properties to genome-wide predictions. Nat. Rev. Genet. 15, 272–286 (2014).

    CAS  Google Scholar 

  9. 9.

    Beagrie, R. A. & Pombo, A. Gene activation by metazoan enhancers: diverse mechanisms stimulate distinct steps of transcription. BioEssays 38, 881–893 (2016).

    CAS  PubMed  Google Scholar 

  10. 10.

    Lenhard, B., Sandelin, A. & Carninci, P. Metazoan promoters: emerging characteristics and insights into transcriptional regulation. Nat. Rev. Genet. 13, 233–245 (2012).

    CAS  PubMed  Google Scholar 

  11. 11.

    Core, L. J. et al. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers. Nat. Genet. 46, 1311–1320 (2014). This study demonstrates a unified divergent architecture of candidate enhancers and promoters and observes a correlation between H3K4me3 and transcription levels.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Andersson, R. et al. Nuclear stability and transcriptional directionality separate functionally distinct RNA species. Nat. Commun. 5, 5336 (2014).

    CAS  PubMed  Google Scholar 

  13. 13.

    Scruggs, B. S. et al. Bidirectional transcription arises from two distinct hubs of transcription factor binding and active chromatin. Mol. Cell 58, 1101–1112 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Chen, Y. et al. Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters. Nat. Genet. 48, 984–994 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Koch, F. et al. Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters. Nat. Struct. Mol. Biol. 18, 956–963 (2011).

    CAS  PubMed  Google Scholar 

  16. 16.

    Andersson, R. et al. Human gene promoters are intrinsically bidirectional. Mol. Cell 60, 346–347 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Diao, Y. et al. A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells. Nat. Methods 14, 629–635 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Dao, L. T. M. et al. Genome-wide characterization of mammalian promoters with distal enhancer functions. Nat. Genet. 49, 1073–1081 (2017).

    CAS  PubMed  Google Scholar 

  19. 19.

    Rajagopal, N. et al. High-throughput mapping of regulatory DNA. Nat. Biotechnol. 34, 167–174 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Engreitz, J. M. et al. Local regulation of gene expression by lncRNA promoters, transcription and splicing. Nature 539, 452–455 (2016). Diao (2017), Dao (2017) and Engreitz (2016) have observed, using in vivo genome editing and in vitro MPRAs, gene promoters with high enhancer strengths.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    De Santa, F. et al. A large fraction of extragenic RNA pol II transcription sites overlap enhancers. PLOS Biol. 8, e1000384 (2010).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Kim, T.-K. et al. Widespread transcription at neuronal activity-regulated enhancers. Nature 465, 182–187 (2010). De Santa (2010) and Kim (2010) are the first studies to observe bidirectional transcription of eRNAs from candidate enhancers at large scale.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Roeder, R. G. The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem. Sci. 21, 327–335 (1996).

    CAS  PubMed  Google Scholar 

  24. 24.

    Smale, S. T. & Kadonaga, J. T. The RNA polymerase II core promoter. Annu. Rev. Biochem. 72, 449–479 (2003).

    CAS  PubMed  Google Scholar 

  25. 25.

    Kadonaga, J. T. Perspectives on the RNA polymerase II core promoter. Wiley Interdiscip. Rev. Dev. Biol. 1, 40–51 (2012).

    CAS  PubMed  Google Scholar 

  26. 26.

    Breathnach, R. & Chambon, P. Organization and expression of eucaryotic split genes coding for proteins. Annu. Rev. Biochem. 50, 349–383 (1981).

    CAS  PubMed  Google Scholar 

  27. 27.

    Carninci, P. et al. Genome-wide analysis of mammalian promoter architecture and evolution. Nat. Genet. 38, 626–635 (2006).

    CAS  PubMed  Google Scholar 

  28. 28.

    Orphanides, G., Lagrange, T. & Reinberg, D. The general transcription factors of RNA polymerase II. Genes Dev. 10, 2657–2683 (1996).

    CAS  PubMed  Google Scholar 

  29. 29.

    Dynlacht, B. D., Hoey, T. & Tjian, R. Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation. Cell 66, 563–576 (1991).

    CAS  PubMed  Google Scholar 

  30. 30.

    Vaquerizas, J. M., Kummerfeld, S. K., Teichmann, S. A. & Luscombe, N. M. A census of human transcription factors: function, expression and evolution. Nat. Rev. Genet. 10, 252–263 (2009).

    CAS  PubMed  Google Scholar 

  31. 31.

    Lambert, S. A. et al. The human transcription factors. Cell 175, 598–599 (2018).

    CAS  PubMed  Google Scholar 

  32. 32.

    Vo, N. & Goodman, R. H. CREB-binding protein and p300 in transcriptional regulation. J. Biol. Chem. 276, 13505–13508 (2001).

    CAS  PubMed  Google Scholar 

  33. 33.

    Malik, S. & Roeder, R. G. Dynamic regulation of pol II transcription by the mammalian mediator complex. Trends Biochem. Sci. 30, 256–263 (2005).

    CAS  PubMed  Google Scholar 

  34. 34.

    Koutelou, E., Hirsch, C. L. & Dent, S. Y. R. Multiple faces of the SAGA complex. Curr. Opin. Cell Biol. 22, 374–382 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Spitz, F. & Furlong, E. E. M. Transcription factors: from enhancer binding to developmental control. Nat. Rev. Genet. 13, 613–626 (2012).

    CAS  PubMed  Google Scholar 

  36. 36.

    Wasserman, W. W. & Sandelin, A. Applied bioinformatics for the identification of regulatory elements. Nat. Rev. Genet. 5, 276–287 (2004).

    CAS  PubMed  Google Scholar 

  37. 37.

    Lettice, L. A. et al. A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum. Mol. Genet. 12, 1725–1735 (2003).

    CAS  PubMed  Google Scholar 

  38. 38.

    Schoenfelder, S. & Fraser, P. Long-range enhancer-promoter contacts in gene expression control. Nat. Rev. Genet. 20, 437–455 (2019).

    CAS  PubMed  Google Scholar 

  39. 39.

    Robson, M. I., Ringel, A. R. & Mundlos, S. Regulatory landscaping: how enhancer-promoter communication is sculpted in 3D. Mol. Cell 74, 1110–1122 (2019).

    CAS  PubMed  Google Scholar 

  40. 40.

    Moreau, P. et al. The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants. Nucleic Acids Res. 9, 6047–6068 (1981).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Banerji, J., Rusconi, S. & Schaffner, W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell 27, 299–308 (1981). Moreau (1981) and Banerji (1981) are the first studies to discover enhancers through the identification of a 72-bp repeat sequence that could enhance gene transcription in SV40. They further show that such enhancers can act both upstream and downstream of, and at various distances from, a reporter gene.

    CAS  PubMed  Google Scholar 

  42. 42.

    Benoist, C. & Chambon, P. In vivo sequence requirements of the SV40 early promotor region. Nature 290, 304–310 (1981).

    CAS  PubMed  Google Scholar 

  43. 43.

    Banerji, J., Olson, L. & Schaffner, W. A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell 33, 729–740 (1983).

    CAS  PubMed  Google Scholar 

  44. 44.

    Neuberger, M. S. Expression and regulation of immunoglobulin heavy chain gene transfected into lymphoid cells. EMBO J. 2, 1373–1378 (1983).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Schaffner, W. Enhancers, enhancers — from their discovery to today’s universe of transcription enhancers. Biol. Chem. 396, 311–327 (2015).

    CAS  PubMed  Google Scholar 

  46. 46.

    Furlong, E. E. M. & Levine, M. Developmental enhancers and chromosome topology. Science 361, 1341–1345 (2018).

    CAS  PubMed  Google Scholar 

  47. 47.

    Catarino, R. R. & Stark, A. Assessing sufficiency and necessity of enhancer activities for gene expression and the mechanisms of transcription activation. Genes Dev. 32, 202–223 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Forrest, A. R. R. et al. A promoter-level mammalian expression atlas. Nature 507, 462–470 (2014).

    CAS  PubMed  Google Scholar 

  49. 49.

    Consortium, R. E. et al. Integrative analysis of 111 reference human epigenomes. Nature 518, 317–330 (2015).

    Google Scholar 

  50. 50.

    Bernstein, B. E. et al. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

    Google Scholar 

  51. 51.

    Arner, E. et al. Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells. Science 347, 1010–1014 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Zavolan, M., van Nimwegen, E. & Gaasterland, T. Splice variation in mouse full-length cDNAs identified by mapping to the mouse genome. Genome Res. 12, 1377–1385 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Landry, J.-R., Mager, D. L. & Wilhelm, B. T. Complex controls: the role of alternative promoters in mammalian genomes. Trends Genet. 19, 640–648 (2003).

    CAS  PubMed  Google Scholar 

  54. 54.

    Valen, E. et al. Genome-wide detection and analysis of hippocampus core promoters using DeepCAGE. Genome Res. 19, 255–265 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Core, L. J., Waterfall, J. J. & Lis, J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science 322, 1845–1848 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Core, L. & Adelman, K. Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation. Genes Dev. 33, 960–982 (2019).

    CAS  PubMed  Google Scholar 

  57. 57.

    Seila, A. C. et al. Divergent transcription from active promoters. Science 322, 1849–1851 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Preker, P. et al. RNA exosome depletion reveals transcription upstream of active human promoters. Science 322, 1851–1854 (2008). Core (2008), Seila (2008) and Preker (2008) are the first studies to independently observe divergent transcription from mammalian gene promoters.

    CAS  PubMed  Google Scholar 

  59. 59.

    Jensen, T. H., Jacquier, A. & Libri, D. Dealing with pervasive transcription. Mol. Cell 52, 473–484 (2013).

    CAS  PubMed  Google Scholar 

  60. 60.

    Seila, A. C., Core, L. J., Lis, J. T. & Sharp, P. A. Divergent transcription: a new feature of active promoters. Cell Cycle 8, 2557–2564 (2009).

    CAS  PubMed  Google Scholar 

  61. 61.

    Ntini, E. et al. Polyadenylation site-induced decay of upstream transcripts enforces promoter directionality. Nat. Struct. Mol. Biol. 20, 923–928 (2013).

    CAS  PubMed  Google Scholar 

  62. 62.

    Almada, A. E., Wu, X., Kriz, A. J., Burge, C. B. & Sharp, P. A. Promoter directionality is controlled by U1 snRNP and polyadenylation signals. Nature 499, 360–363 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Furey, T. S. ChIP-seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions. Nat. Rev. Genet. 13, 840–852 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Bernstein, B. E. et al. Methylation of histone H3 Lys 4 in coding regions of active genes. Proc. Natl Acad. Sci. USA 99, 8695–8700 (2002).

    CAS  PubMed  Google Scholar 

  65. 65.

    Lee, C. K., Shibata, Y., Rao, B., Strahl, B. D. & Lieb, J. D. Evidence for nucleosome depletion at active regulatory regions genome-wide. Nat. Genet. 36, 900–905 (2004).

    CAS  PubMed  Google Scholar 

  66. 66.

    Schübeler, D. et al. The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote. Genes Dev. 18, 1263–1271 (2004).

    PubMed  PubMed Central  Google Scholar 

  67. 67.

    Roh, T., Ngau, W. C., Cui, K., Landsman, D. & Zhao, K. High-resolution genome-wide mapping of histone modifications. Nat. Biotechnol. 22, 1013–1016 (2004).

    CAS  PubMed  Google Scholar 

  68. 68.

    Song, L. et al. Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity. Genome Res. 21, 1757–1767 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Kim, T. H. et al. A high-resolution map of active promoters in the human genome. Nature 436, 876–880 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 70.

    Zeitlinger, J. et al. RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo. Nat. Genet. 39, 1512–1516 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. 71.

    Muse, G. W. et al. RNA polymerase is poised for activation across the genome. Nat. Genet. 39, 1507–1511 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. 72.

    Klemm, S. L., Shipony, Z. & Greenleaf, W. J. Chromatin accessibility and the regulatory epigenome. Nat. Rev. Genet. 20, 207–220 (2019).

    CAS  PubMed  Google Scholar 

  73. 73.

    Creyghton, M. P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl Acad. Sci. USA 107, 21931–21936 (2010).

    CAS  PubMed  Google Scholar 

  74. 74.

    Heintzman, N. D. et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 39, 311–318 (2007).

    CAS  Google Scholar 

  75. 75.

    Robertson, A. G. et al. Genome-wide relationship between histone H3 lysine 4 mono- and tri-methylation and transcription factor binding. Genome Res. 18, 1906–1917 (2008). Heintzman (2007) and Robertson (2008) identify a signature of high H3K4me1 and low H3K4me3 at candidate enhancers that could be used to discern enhancers from promoters and predict their locations genome-wide.

    CAS  PubMed  PubMed Central  Google Scholar 

  76. 76.

    Ernst, J. et al. Mapping and analysis of chromatin state dynamics in nine human cell types. Nature 473, 43–49 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. 77.

    Hoffman, M. M. et al. Unsupervised pattern discovery in human chromatin structure through genomic segmentation. Nat. Methods 9, 473–476 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. 78.

    Ernst, J. & Kellis, M. ChromHMM: automating chromatin-state discovery and characterization. Nat. Methods 9, 215–216 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. 79.

    Melgar, M. F., Collins, F. S. & Sethupathy, P. Discovery of active enhancers through bidirectional expression of short transcripts. Genome Biol. 12, R113 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. 80.

    Wu, H. et al. Tissue-specific RNA expression marks distant-acting developmental enhancers. PLOS Genet. 10, e1004610 (2014).

    PubMed  PubMed Central  Google Scholar 

  81. 81.

    Andersson, R., Sandelin, A. & Danko, C. G. A unified architecture of transcriptional regulatory elements. Trends Genet. 31, 426–433 (2015).

    CAS  PubMed  Google Scholar 

  82. 82.

    van Arensbergen, J. et al. Genome-wide mapping of autonomous promoter activity in human cells. Nat. Biotechnol. 35, 145–153 (2017).

    PubMed  Google Scholar 

  83. 83.

    Landolin, J. M. et al. Sequence features that drive human promoter function and tissue specificity. Genome Res. 20, 890–898 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. 84.

    Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. 85.

    Soares, L. M. et al. Determinants of histone H3K4 methylation patterns. Mol. Cell 68, 773–785.e6 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. 86.

    Pekowska, A. et al. H3K4 tri-methylation provides an epigenetic signature of active enhancers. EMBO J. 30, 4198–4210 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. 87.

    Henriques, T. et al. Widespread transcriptional pausing and elongation control at enhancers. Genes Dev. 32, 26–41 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. 88.

    Rennie, S. et al. Transcription start site analysis reveals widespread divergent transcription in D. melanogaster and core promoter-encoded enhancer activities. Nucleic Acids Res. 46, 5455–5469 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. 89.

    Mifsud, B. et al. Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C. Nat. Genet. 47, 598–606 (2015).

    CAS  PubMed  Google Scholar 

  90. 90.

    Schoenfelder, S. et al. The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements. Genome Res. 25, 582–597 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. 91.

    Javierre, B. M. et al. Lineage-specific genome architecture links enhancers and non-coding disease variants to target gene promoters. Cell 167, 1369–1384.e19 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. 92.

    Xu, Z., Wei, G., Chepelev, I., Zhao, K. & Felsenfeld, G. Mapping of INS promoter interactions reveals its role in long-range regulation of SYT8 transcription. Nat. Struct. Mol. Biol. 18, 372–378 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. 93.

    Andersson, R. Promoter or enhancer, what’s the difference? Deconstruction of established distinctions and presentation of a unifying model. BioEssays 37, 314–323 (2015).

    PubMed  Google Scholar 

  94. 94.

    Mikhaylichenko, O. et al. The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription. Genes Dev. 32, 42–57 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. 95.

    Canver, M. C. et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  96. 96.

    Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR–Cas9. Nat. Biotechnol. 34, 192–198 (2016).

    CAS  PubMed  Google Scholar 

  97. 97.

    Sanjana, N. E. et al. High-resolution interrogation of functional elements in the noncoding genome. Science 353, 1545–1549 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  98. 98.

    Klann, T. S. et al. CRISPR–Cas9 epigenome editing enables high-throughput screening for functional regulatory elements in the human genome. Nat. Biotechnol. 35, 561–568 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  99. 99.

    Carleton, J. B., Berrett, K. C. & Gertz, J. Multiplex enhancer interference reveals collaborative control of gene regulation by estrogen receptor α-bound enhancers. Cell Syst. 5, 333–344.e5 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  100. 100.

    Simeonov, D. R. et al. Discovery of stimulation-responsive immune enhancers with CRISPR activation. Nature 549, 111–115 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  101. 101.

    Nguyen, T. A. et al. High-throughput functional comparison of promoter and enhancer activities. Genome Res. 26, 1023–1033 (2016). This is the first study to systematically compare the promoter and enhancer potentials of a large number of candidate sequences using MPRAs.

    CAS  PubMed  PubMed Central  Google Scholar 

  102. 102.

    Weingarten-Gabbay, S. et al. Systematic interrogation of human promoters. Genome Res. 29, 171–183 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. 103.

    Mattioli, K. et al. High-throughput functional analysis of lncRNA core promoters elucidates rules governing tissue specificity. Genome Res. 29, 344–355 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  104. 104.

    Patwardhan, R. P. et al. Massively parallel functional dissection of mammalian enhancers in vivo. Nat. Biotechnol. 30, 265–270 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. 105.

    Melnikov, A. et al. Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Nat. Biotechnol. 30, 271–277 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. 106.

    Kheradpour, P. et al. Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay. Genome Res. 23, 800–811 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  107. 107.

    Kwasnieski, J. C., Mogno, I., Myers, C. A., Corbo, J. C. & Cohen, B. A. Complex effects of nucleotide variants in a mammalian cis-regulatory element. Proc. Natl Acad. Sci. USA 109, 19498–19503 (2012).

    CAS  PubMed  Google Scholar 

  108. 108.

    Smith, R. P. et al. Massively parallel decoding of mammalian regulatory sequences supports a flexible organizational model. Nat. Genet. 45, 1021–1028 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  109. 109.

    Grossman, S. R. et al. Systematic dissection of genomic features determining transcription factor binding and enhancer function. Proc. Natl Acad. Sci. USA 114, E1291–E1300 (2017).

    CAS  PubMed  Google Scholar 

  110. 110.

    Shen, S. Q. et al. Massively parallel cis-regulatory analysis in the mammalian central nervous system. Genome Res. 26, 238–255 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  111. 111.

    Haberle, V. et al. Transcriptional cofactors display specificity for distinct types of core promoters. Nature 570, 122–126 (2019).

    CAS  PubMed  Google Scholar 

  112. 112.

    Kwasnieski, J. C., Fiore, C., Chaudhari, H. G. & Cohen, B. A. High-throughput functional testing of ENCODE segmentation predictions. Genome Res. 24, 1595–1602 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. 113.

    Chaudhari, H. G. & Cohen, B. A. Local sequence features that influence AP-1-regulatory activity. Genome Res. 28, 171–181 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  114. 114.

    Muerdter, F. et al. Resolving systematic errors in widely used enhancer activity assays in human cells. Nat. Methods 15, 141–149 (2018).

    CAS  PubMed  Google Scholar 

  115. 115.

    Fenouil, R. et al. CpG islands and GC content dictate nucleosome depletion in a transcription-independent manner at mammalian promoters. Genome Res. 22, 2399–2408 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  116. 116.

    Biddie, S. C. et al. Transcription factor AP1 potentiates chromatin accessibility and glucocorticoid receptor binding. Mol. Cell 43, 145–155 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  117. 117.

    Iwafuchi-Doi, M. & Zaret, K. S. Pioneer transcription factors in cell reprogramming. Genes Dev. 28, 2679–2692 (2014).

    PubMed  PubMed Central  Google Scholar 

  118. 118.

    Nakayama, R. T. et al. SMARCB1 is required for widespread BAF complex-mediated activation of enhancers and bivalent promoters. Nat. Genet. 49, 1613–1623 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  119. 119.

    Colbran, L. L., Chen, L. & Capra, J. A. Sequence characteristics distinguish transcribed enhancers from promoters and predict their breadth of activity. Genetics 211, 1205–1217 (2019).

    CAS  PubMed  Google Scholar 

  120. 120.

    Farley, E. K. et al. Suboptimization of developmental enhancers. Science 350, 325–328 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  121. 121.

    Farley, E. K., Olson, K. M., Zhang, W., Rokhsar, D. S. & Levine, M. S. Syntax compensates for poor binding sites to encode tissue specificity of developmental enhancers. Proc. Natl Acad. Sci. USA 113, 6508–6513 (2016).

    CAS  PubMed  Google Scholar 

  122. 122.

    Cheng, Y. et al. Principles of regulatory information conservation between mouse and human. Nature 515, 371–375 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. 123.

    Kim, T.-K. & Shiekhattar, R. Architectural and functional commonalities between enhancers and promoters. Cell 162, 948–959 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. 124.

    Struhl, K. Transcriptional noise and the fidelity of initiation by RNA polymerase II. Nat. Struct. Mol. Biol. 14, 103–105 (2007).

    CAS  PubMed  Google Scholar 

  125. 125.

    Zabidi, M. A. et al. Enhancer–core–promoter specificity separates developmental and housekeeping gene regulation. Nature 518, 556–559 (2015).

    CAS  PubMed  Google Scholar 

  126. 126.

    Dickel, D. E. et al. Ultraconserved enhancers are required for normal development. Cell 172, 491–499.e15 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. 127.

    Pennacchio, L. A. et al. In vivo enhancer analysis of human conserved non-coding sequences. Nature 444, 499–502 (2006).

    CAS  PubMed  Google Scholar 

  128. 128.

    Kidwell, M. & Lisch, D. Transposable elements and host genome evolution. Trends Ecol. Evol. 15, 95–99 (2000).

    CAS  PubMed  Google Scholar 

  129. 129.

    Wu, X. & Sharp, P. A. Divergent transcription: a driving force for new gene origination? Cell 155, 990–996 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  130. 130.

    Carelli, F. N., Liechti, A., Halbert, J., Warnefors, M. & Kaessmann, H. Repurposing of promoters and enhancers during mammalian evolution. Nat. Commun. 9, 4066 (2018).

    PubMed  PubMed Central  Google Scholar 

  131. 131.

    Halfon, M. S. Studying transcriptional enhancers: the founder fallacy, validation creep, and other biases. Trends Genet. 35, 93–103 (2019).

    CAS  PubMed  Google Scholar 

  132. 132.

    Ramirez-Carrozzi, V. R. et al. A unifying model for the selective regulation of inducible transcription by CpG islands and nucleosome remodeling. Cell 138, 114–128 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  133. 133.

    Kolovos, P., Knoch, T. A., Grosveld, F. G., Cook, P. R. & Papantonis, A. Enhancers and silencers: an integrated and simple model for their function. Epigenetics Chromatin 5, 1 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  134. 134.

    Sima, J. et al. Identifying cis elements for spatiotemporal control of mammalian DNA replication. Cell 176, 816–830.e18 (2019).

    CAS  PubMed  Google Scholar 

  135. 135.

    Kawai, J. et al. Functional annotation of a full-length mouse cDNA collection. Nature 409, 685–690 (2001).

    PubMed  Google Scholar 

  136. 136.

    Takahashi, H., Lassmann, T., Murata, M. & Carninci, P. 5′ end-centered expression profiling using cap-analysis gene expression and next-generation sequencing. Nat. Protoc. 7, 542–561 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  137. 137.

    Yamashita, R. et al. Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis. Genome Res. 21, 775–789 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  138. 138.

    Kwak, H., Fuda, N. J., Core, L. J. & Lis, J. T. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing. Science 339, 950–953 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  139. 139.

    Kruesi, W. S., Core, L. J., Waters, C. T., Lis, J. T. & Meyer, B. J. Condensin controls recruitment of RNA polymerase II to achieve nematode X-chromosome dosage compensation. eLife 2, e00808 (2013).

    PubMed  PubMed Central  Google Scholar 

  140. 140.

    Lam, M. T. Y. et al. Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription. Nature 498, 511–515 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  141. 141.

    Mayer, A. et al. Native elongating transcript sequencing reveals human transcriptional activity at nucleotide resolution. Cell 161, 541–554 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  142. 142.

    Nojima, T. et al. Mammalian NET-seq reveals genome-wide nascent transcription coupled to RNA processing. Cell 161, 526–540 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  143. 143.

    Weber, C. M., Ramachandran, S. & Henikoff, S. Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase. Mol. Cell 53, 819–830 (2014).

    CAS  PubMed  Google Scholar 

  144. 144.

    Nechaev, S. et al. Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila. Science 327, 335–338 (2010).

    CAS  PubMed  Google Scholar 

  145. 145.

    Berman, B. P. et al. Exploiting transcription factor binding site clustering to identify cis-regulatory modules involved in pattern formation in the Drosophila genome. Proc. Natl Acad. Sci. USA 99, 757–762 (2002).

    CAS  PubMed  Google Scholar 

  146. 146.

    May, D. et al. Large-scale discovery of enhancers from human heart tissue. Nat. Genet. 44, 89–93 (2011).

    PubMed  PubMed Central  Google Scholar 

  147. 147.

    Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  148. 148.

    Wang, Z. et al. Combinatorial patterns of histone acetylations and methylations in the human genome. Nat. Genet. 40, 897–903 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  149. 149.

    Thurman, R. E. et al. The accessible chromatin landscape of the human genome. Nature 489, 75–82 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  150. 150.

    Cusanovich, D. A. et al. A single-cell atlas of in vivo mammalian chromatin accessibility. Cell 174, 1309–1324.e18 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  151. 151.

    Pradeepa, M. M. et al. Histone H3 globular domain acetylation identifies a new class of enhancers. Nat. Genet. 48, 681–686 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  152. 152.

    Rajagopal, N. et al. Distinct and predictive histone lysine acetylation patterns at promoters, enhancers, and gene bodies. G3 4, 2051–2063 (2014).

    PubMed  Google Scholar 

  153. 153.

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  154. 154.

    Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  155. 155.

    Fulco, C. P. et al. Systematic mapping of functional enhancer-promoter connections with CRISPR interference. Science 354, 769–773 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  156. 156.

    Matharu, N. et al. CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. Science 363, eaau0629 (2019).

    Article  PubMed  Google Scholar 

  157. 157.

    Lopes, R., Korkmaz, G. & Agami, R. Applying CRISPR–Cas9 tools to identify and characterize transcriptional enhancers. Nat. Rev. Mol. Cell Biol. 17, 597–604 (2016).

    CAS  PubMed  Google Scholar 

  158. 158.

    Patwardhan, R. P. et al. High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis. Nat. Biotechnol. 27, 1173–1175 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  159. 159.

    Inoue, F. et al. A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity. Genome Res. 27, 38–52 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  160. 160.

    Arnold, C. D. et al. Genome-wide quantitative enhancer activity maps identified by STARR-seq. Science 339, 1074–1077 (2013).

    CAS  PubMed  Google Scholar 

  161. 161.

    Arnold, C. D. et al. Genome-wide assessment of sequence-intrinsic enhancer responsiveness at single-base-pair resolution. Nat. Biotechnol. 35, 136–144 (2017).

    CAS  PubMed  Google Scholar 

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Acknowledgements

R.A. was supported by the Independent Research Fund Denmark (6108-00038B), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (StG no. 638173), and the Novo Nordisk Foundation (NNF18OC0052570). A.S. was supported by the ERC under the European Union’s Horizon 2020 research and innovation programme (MSCA ITN pHioniC), the Lundbeck Foundation, the Danish Cancer Society, the Danish Council for Independent Research and Innovation Fund Denmark. We thank L. van Duin, M. Wu and A. Thieffry for critical comments on the manuscript.

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Glossary

RNA polymerase II

(RNAPII). An enzyme that catalyses the transcription of DNA to RNA, including mRNAs and many long non-coding RNAs.

Transcription start site

(TSS). The first transcribed genomic nucleotide of a transcript.

General transcription factors

(GTFs). Proteins that, together with RNA polymerase II, make up the pre-initiation complex.

Pre-initiation complex

A polypeptide complex consisting of RNA polymerase II and general transcription factors. This forms around the transcription start site and primes RNA polymerase II for transcription.

TATA box

A T/A-rich sequence that lies upstream (typically 24–30 bp) of transcription start sites, with a role in positioning the pre-initiation complex.

INR

The initiator sequence; a sequence pattern often overlapping transcription start sites.

Transcription factors

(TFs). Sequence-specific DNA-binding proteins with a role in regulating transcription.

Nucleosome-depleted region

(NDR). A region depleted of nucleosomes, often identified by DNaseI hypersensitivity, and often carrying regulatory potential.

Nascent-RNA techniques

A wide range of methods aimed at capturing RNA as it is being transcribed. See Box 2.

DNase-seq

A method for identifying accessible regions of the genome, based on DNaseI hypersensitivity. See Box 3.

Promoter-upstream transcript

(PROMPT). A short RNA (also known as upstream antisense RNA (uaRNA)) that is transcribed upstream and on the opposite strand from an mRNA transcription start site and is typically degraded by the nuclear exosome. It has many similarities to an enhancer RNA.

Nuclear exosome

A multi-protein complex responsible for the degradation of RNAs from the 3′ end.

Poly(A) sites

Sequence patterns (AT/ATAA) associated with the 3′ ends of genes, but that also occur with high frequency in intergenic DNA.

ChIP–seq

Chromatin immunoprecipitation coupled to sequencing; a method for finding DNA–protein interactions by combining immunoprecipitation and high-throughput DNA sequencing. See Box 3.

Enhancer RNAs

(eRNAs). Short RNAs (<500 bp) that are transcribed from enhancers, with many similarities to promoter-upstream transcripts (PROMPTs).

ATAC-seq

Assay for transposase-accessible chromatin; a method for identifying accessible regions of the genome, based on transposase activity.

CpG islands

Genomic sequences that are not depleted of cytosine–phosphate–guanine (CpG) dinucleotides, which would occur by 5-methylcytosine deamination. They often overlap or are near transcription start sites. Most definitions set a minimum length (for example, 200 or 500 bp) and a minimum observed/expected CpG ratio.

CAGE

Cap analysis of gene expression; a method to identify transcription start sites by sequencing the 5′ ends of capped, steady-state RNAs. See Box 2.

Expression quantitative trait loci

(eQTLs). Regions of DNA in which genetic variation is associated with variability in the expression of one or more genes.

Massively parallel reporter assays

(MPRAs). Methods that can measure the promoter or enhancer activity of many candidate DNA sequences in parallel. See Box 5.

DNase hypersensitive sites

(DHSs). Highly accessible genomic regions identified by DNase-seq.

Pioneer transcription factors

Transcription factors that can directly bind nucleosomal DNA, possibly in compacted chromatin.

Homotypic attraction

A force driving chromatin with similar characteristics or associated proteins to self-associate. The physical association may be formed by protein bridges or liquid–liquid phase separation, the latter of which involves molecules separating into liquid condensates with specific compositions.

Ascertainment bias

Drawing general conclusions based on biased sampling of non-representative examples.

Validation creep

Treating predictions as validated entities, in either the same study or subsequent studies. This type of bias is related to ascertainment bias and overfitting.

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Andersson, R., Sandelin, A. Determinants of enhancer and promoter activities of regulatory elements. Nat Rev Genet 21, 71–87 (2020). https://doi.org/10.1038/s41576-019-0173-8

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