Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Identification of non-coding silencer elements and their regulation of gene expression

Abstract

Cell type- and differentiation-specific gene expression is precisely controlled by genomic non-coding regulatory elements (NCREs), which include promoters, enhancers, silencers and insulators. It is estimated that more than 90% of disease-associated sequence variants lie within the non-coding part of the genome, potentially affecting the activity of NCREs. Consequently, the functional annotation of NCREs is a major driver of genome research. Compared with our knowledge of other regulatory elements, our knowledge of silencers, which are NCREs that repress the transcription of genes, is largely lacking. Multiple recent studies have reported large-scale identification of transcription silencer elements, indicating their importance in homeostasis and disease. In this Review, we discuss the biology of silencers, including methods for their discovery, epigenomic and other characteristics, and modes of function of silencers. We also discuss important silencer-relevant considerations in assessing data from genome-wide association studies and shed light on potential future silencer-based therapeutic applications.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Silencer elements and their proposed mechanisms in gene regulation.
Fig. 2: Large-scale silencer identification methods.
Fig. 3: Silencer elements with dual functionality.
Fig. 4: Effects of disrupted silencer function on gene expression and disease.

References

  1. Nurk, S. et al. The complete sequence of a human genome. Science 376, 44–53 (2022).

    Article  Google Scholar 

  2. Jackson, M., Marks, L., May, G. H. W. & Wilson, J. B. The genetic basis of disease. Essays Biochem. 62, 643–723 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  3. ENCODE Project Consortium. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447, 799–816 (2007).

    Article  Google Scholar 

  4. Venter, J. C. et al. The sequence of the human genome. Science 291, 1304–1351 (2001).

    Article  CAS  PubMed  Google Scholar 

  5. Lander, E. S. et al. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001).

    Article  CAS  PubMed  Google Scholar 

  6. Schaub, M. A., Boyle, A. P., Kundaje, A., Batzoglou, S. & Snyder, M. Linking disease associations with regulatory information in the human genome. Genome Res. 22, 1748–1759 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Khurana, E. et al. Role of non-coding sequence variants in cancer. Nat. Rev. Genet. 17, 93–108 (2016).

    Article  CAS  PubMed  Google Scholar 

  8. Dunham, I. et al. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

    Article  CAS  Google Scholar 

  9. Riethoven, J. J. Regulatory regions in DNA: promoters, enhancers, silencers, and insulators. Methods Mol. Biol. 674, 33–42 (2010).

    Article  CAS  PubMed  Google Scholar 

  10. Butler, J. E. & Kadonaga, J. T. The RNA polymerase II core promoter: a key component in the regulation of gene expression. Genes Dev. 16, 2583–2592 (2002).

    Article  CAS  PubMed  Google Scholar 

  11. Kokubu, C. et al. A transposon-based chromosomal engineering method to survey a large cis-regulatory landscape in mice. Nat. Genet. 41, 946–952 (2009).

    Article  CAS  PubMed  Google Scholar 

  12. 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).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  14. Calo, E. & Wysocka, J. Modification of enhancer chromatin: what, how, and why? Mol. Cell 49, 825–837 (2013).

    Article  CAS  PubMed  Google Scholar 

  15. Gao, T. & Qian, J. EnhancerAtlas 2.0: an updated resource with enhancer annotation in 586 tissue/cell types across nine species. Nucleic Acids Res. 48, D58–D64 (2020).

    CAS  PubMed  Google Scholar 

  16. Visel, A., Minovitsky, S., Dubchak, I. & Pennacchio, L. A. VISTA Enhancer Browser — a database of tissue-specific human enhancers. Nucleic Acids Res. 35, D88–D92 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. de Laat, W. & Duboule, D. Topology of mammalian developmental enhancers and their regulatory landscapes. Nature 502, 499–506 (2013).

    Article  PubMed  Google Scholar 

  18. Zheng, H. & Xie, W. The role of 3D genome organization in development and cell differentiation. Nat. Rev. Mol. Cell Biol. 20, 535–550 (2019).

    Article  CAS  PubMed  Google Scholar 

  19. Rowley, M. J. & Corces, V. G. Organizational principles of 3D genome architecture. Nat. Rev. Genet. 19, 789–800 (2018).

    Article  CAS  PubMed  Google Scholar 

  20. Bushey, A. M., Dorman, E. R. & Corces, V. G. Chromatin insulators: regulatory mechanisms and epigenetic inheritance. Mol. Cell 32, 1–9 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ali, T., Renkawitz, R. & Bartkuhn, M. Insulators and domains of gene expression. Curr. Opin. Genet. Dev. 37, 17–26 (2016).

    Article  CAS  PubMed  Google Scholar 

  22. Özdemir, I. & Gambetta, M. C. The role of insulation in patterning gene expression. Genes 10, 767 (2019).

    Article  PubMed Central  Google Scholar 

  23. Brasset, E. & Vaury, C. Insulators are fundamental components of the eukaryotic genomes. Heredity 94, 571–576 (2005).

    Article  CAS  PubMed  Google Scholar 

  24. Allshire, R. C. & Madhani, H. D. Ten principles of heterochromatin formation and function. Nat. Rev. Mol. Cell Biol. 19, 229–244 (2018).

    Article  CAS  PubMed  Google Scholar 

  25. Ogbourne, S. & Antalis, T. M. Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes. Biochem. J. 331, 1–14 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Courey, A. J. & Jia, S. Transcriptional repression: the long and the short of it. Genes Dev. 15, 2786–2796 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Friedman, R. Z. et al. Information content differentiates enhancers from silencers in mouse photoreceptors. Elife 10, e67403 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Gaston, K. & Jayaraman, P. S. Transcriptional repression in eukaryotes: repressors and repression mechanisms. Cell Mol. Life Sci. 60, 721–741 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Abraham, J., Nasmyth, K. A., Strathern, J. N., Klar, A. J. & Hicks, J. B. Regulation of mating-type information in yeast. Negative control requiring sequences both 5′ and 3′ to the regulated region. J. Mol. Biol. 176, 307–331 (1984).

    Article  CAS  PubMed  Google Scholar 

  30. Brand, A. H., Breeden, L., Abraham, J., Sternglanz, R. & Nasmyth, K. Characterization of a “silencer” in yeast: a DNA sequence with properties opposite to those of a transcriptional enhancer. Cell 41, 41–48 (1985).

    Article  CAS  PubMed  Google Scholar 

  31. Jiang, J., Cai, H., Zhou, Q. & Levine, M. Conversion of a dorsal-dependent silencer into an enhancer: evidence for dorsal corepressors. EMBO J. 12, 3201–3209 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Siu, G., Wurster, A. L., Duncan, D. D., Soliman, T. M. & Hedrick, S. M. A transcriptional silencer controls the developmental expression of the CD4 gene. EMBO J. 13, 3570–3579 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sawada, S., Scarborough, J. D., Killeen, N. & Littman, D. R. A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development. Cell 77, 917–929 (1994).

    Article  CAS  PubMed  Google Scholar 

  34. Donda, A., Schulz, M., Bürki, K., De Libero, G. & Uematsu, Y. Identification and characterization of a human CD4 silencer. Eur. J. Immunol. 26, 493–500 (1996).

    Article  CAS  PubMed  Google Scholar 

  35. Taniuchi, I., Sunshine, M. J., Festenstein, R. & Littman, D. R. Evidence for distinct CD4 silencer functions at different stages of thymocyte differentiation. Mol. Cell 10, 1083–1096 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Mori, N., Stein, R., Sigmund, O. & Anderson, D. J. A cell type-preferred silencer element that controls the neural-specific expression of the SCG10 gene. Neuron 4, 583–594 (1990).

    Article  CAS  PubMed  Google Scholar 

  37. Bruce, A. W. et al. Genome-wide analysis of repressor element 1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) target genes. Proc. Natl Acad. Sci. USA 101, 10458–10463 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Mortazavi, A., Leeper Thompson, E. C., Garcia, S. T., Myers, R. M. & Wold, B. Comparative genomics modeling of the NRSF/REST repressor network: from single conserved sites to genome-wide repertoire. Genome Res. 16, 1208–1221 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Johnson, R. et al. Identification of the REST regulon reveals extensive transposable element-mediated binding site duplication. Nucleic Acids Res. 34, 3862–3877 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Chong, J. A. et al. REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons. Cell 80, 949–957 (1995).

    Article  CAS  PubMed  Google Scholar 

  41. Schoenherr, C. J. & Anderson, D. J. The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes. Science 267, 1360–1363 (1995).

    Article  CAS  PubMed  Google Scholar 

  42. Anderson, D. J. & Axel, R. Molecular probes for the development and plasticity of neural crest derivatives. Cell 42, 649–662 (1985).

    Article  CAS  PubMed  Google Scholar 

  43. Mori, N., Schoenherr, C., Vandenbergh, D. J. & Anderson, D. J. A common silencer element in the SCG10 and type II Na+ channel genes binds a factor present in nonneuronal cells but not in neuronal cells. Neuron 9, 45–54 (1992).

    Article  CAS  PubMed  Google Scholar 

  44. Petrykowska, H. M., Vockley, C. M. & Elnitski, L. Detection and characterization of silencers and enhancer-blockers in the greater CFTR locus. Genome Res. 18, 1238–1246 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Gisselbrecht, S. S. et al. Transcriptional silencers in Drosophila serve a dual role as transcriptional enhancers in alternate cellular contexts. Mol. Cell 77, 324–337.e328 (2020).

    Article  CAS  PubMed  Google Scholar 

  46. Cassandri, M. et al. Zinc-finger proteins in health and disease. Cell Death Discov. 3, 17071 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Lupo, A. et al. KRAB-zinc finger proteins: a repressor family displaying multiple biological functions. Curr. Genom. 14, 268–278 (2013).

    Article  CAS  Google Scholar 

  48. Ecco, G., Imbeault, M. & Trono, D. KRAB zinc finger proteins. Development 144, 2719–2729 (2017).

    Article  CAS  PubMed  Google Scholar 

  49. Urrutia, R. KRAB-containing zinc-finger repressor proteins. Genome Biol. 4, 231 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Klochkov, D. et al. A CTCF-dependent silencer located in the differentially methylated area may regulate expression of a housekeeping gene overlapping a tissue-specific gene domain. Mol. Cell Biol. 26, 1589–1597 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Ye, J., Cippitelli, M., Dorman, L., Ortaldo, J. R. & Young, H. A. The nuclear factor YY1 suppresses the human gamma interferon promoter through two mechanisms: inhibition of AP1 binding and activation of a silencer element. Mol. Cell Biol. 16, 4744–4753 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Zhang, X., Diab, I. H. & Zehner, Z. E. ZBP-89 represses vimentin gene transcription by interacting with the transcriptional activator, Sp1. Nucleic Acids Res. 31, 2900–2914 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kuo, M. H. & Allis, C. D. Roles of histone acetyltransferases and deacetylases in gene regulation. Bioessays 20, 615–626 (1998).

    Article  CAS  PubMed  Google Scholar 

  54. Berger, S. L. Histone modifications in transcriptional regulation. Curr. Opin. Genet. Dev. 12, 142–148 (2002).

    Article  CAS  PubMed  Google Scholar 

  55. Heintzman, N. D. et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Huang, D., Petrykowska, H. M., Miller, B. F., Elnitski, L. & Ovcharenko, I. Identification of human silencers by correlating cross-tissue epigenetic profiles and gene expression. Genome Res. 29, 657–667 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Segert, J. A., Gisselbrecht, S. S. & Bulyk, M. L. Transcriptional silencers: driving gene expression with the brakes on. Trends Genet. 37, 514–527 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Zhang, Y., See, Y. X., Tergaonkar, V. & Fullwood, M. J. Long-distance repression by human silencers: chromatin interactions and phase separation in silencers. Cells 11, 1560 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  60. Doni Jayavelu, N., Jajodia, A., Mishra, A. & Hawkins, R. D. Candidate silencer elements for the human and mouse genomes. Nat. Commun. 11, 1061 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Cornejo-Páramo, P., Roper, K., Degnan, S. M., Degnan, B. M. & Wong, E. S. Distal regulation, silencers, and a shared combinatorial syntax are hallmarks of animal embryogenesis. Genome Res. 32, 474–487 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Pang, B. & Snyder, M. P. Systematic identification of silencers in human cells. Nat. Genet. 52, 254–263 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Hansen, T. J. & Hodges, E. ATAC-STARR-seq reveals transcription factor-bound activators and silencers across the chromatin accessible human genome. Genome Res. 32, 1529–1541 (2022).

    Article  PubMed Central  Google Scholar 

  64. Cai, Y. et al. H3K27me3-rich genomic regions can function as silencers to repress gene expression via chromatin interactions. Nat. Commun. 12, 719 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Piunti, A. & Shilatifard, A. The roles of Polycomb repressive complexes in mammalian development and cancer. Nat. Rev. Mol. Cell Biol. 22, 326–345 (2021).

    Article  CAS  PubMed  Google Scholar 

  66. Blackledge, N. P. & Klose, R. J. The molecular principles of gene regulation by Polycomb repressive complexes. Nat. Rev. Mol. Cell Biol. 22, 815–833 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Grossniklaus, U. & Paro, R. Transcriptional silencing by Polycomb-group proteins. Cold Spring Harb. Perspect. Biol. 6, a019331 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  68. Ogiyama, Y., Schuettengruber, B., Papadopoulos, G. L., Chang, J. M. & Cavalli, G. Polycomb-dependent chromatin looping contributes to gene silencing during Drosophila development. Mol. Cell 71, 73–88.e75 (2018).

    Article  CAS  PubMed  Google Scholar 

  69. Bantignies, F. et al. Polycomb-dependent regulatory contacts between distant Hox loci in Drosophila. Cell 144, 214–226 (2011).

    Article  CAS  PubMed  Google Scholar 

  70. Tiwari, V. K. et al. PcG proteins, DNA methylation, and gene repression by chromatin looping. PLoS Biol. 6, 2911–2927 (2008).

    Article  CAS  PubMed  Google Scholar 

  71. Ngan, C. Y. et al. Chromatin interaction analyses elucidate the roles of PRC2-bound silencers in mouse development. Nat. Genet. 52, 264–272 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Akinci, E., Hamilton, M. C., Khowpinitchai, B. & Sherwood, R. I. Using CRISPR to understand and manipulate gene regulation. Development 148, dev182667 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Adli, M. The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9, 1911 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  74. Tycko, J. et al. Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements. Nat. Commun. 10, 4063 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Tarjan, D. R., Flavahan, W. A. & Bernstein, B. E. Epigenome editing strategies for the functional annotation of CTCF insulators. Nat. Commun. 10, 4258 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  79. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Gasperini, M., Tome, J. M. & Shendure, J. Towards a comprehensive catalogue of validated and target-linked human enhancers. Nat. Rev. Genet. 21, 292–310 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. van Arensbergen, J., van Steensel, B. & Bussemaker, H. J. In search of the determinants of enhancer–promoter interaction specificity. Trends Cell Biol. 24, 695–702 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  82. Martinez-Ara, M., Comoglio, F., van Arensbergen, J. & van Steensel, B. Systematic analysis of intrinsic enhancer-promoter compatibility in the mouse genome. Mol. Cell 82, 2519–2531 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Cheng, C. K., Wong, T. H. Y., Yung, Y. L., Chan, N. C. N. & Ng, M. H. L. Investigation of the transcriptional role of a RUNX1 intronic silencer by CRISPR/Cas9 ribonucleoprotein in acute myeloid leukemia cells. J. Vis. Exp. https://doi.org/10.3791/60130 (2019).

    Article  PubMed  Google Scholar 

  84. Young, M. D. et al. ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity. Nucleic Acids Res. 39, 7415–7427 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Hosogane, M., Funayama, R., Shirota, M. & Nakayama, K. Lack of transcription triggers H3K27me3 accumulation in the gene body. Cell Rep. 16, 696–706 (2016).

    Article  CAS  PubMed  Google Scholar 

  86. Margueron, R. & Reinberg, D. The Polycomb complex PRC2 and its mark in life. Nature 469, 343–349 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Czermin, B. et al. Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites. Cell 111, 185–196 (2002).

    Article  CAS  PubMed  Google Scholar 

  88. Shen, X. et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol. Cell 32, 491–502 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Lee, S. W., Oh, Y. M., Lu, Y. L., Kim, W. K. & Yoo, A. S. MicroRNAs overcome cell fate barrier by reducing EZH2-controlled REST stability during neuronal conversion of human adult fibroblasts. Dev. Cell 46, 73–84.e77 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Jepsen, K. et al. Combinatorial roles of the nuclear receptor corepressor in transcription and development. Cell 102, 753–763 (2000).

    Article  CAS  PubMed  Google Scholar 

  91. Beck, D. B., Oda, H., Shen, S. S. & Reinberg, D. PR-Set7 and H4K20me1: at the crossroads of genome integrity, cell cycle, chromosome condensation, and transcription. Genes Dev. 26, 325–337 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. van Nuland, R. & Gozani, O. Histone H4 lysine 20 (H4K20) methylation, expanding the signaling potential of the proteome one methyl moiety at a time. Mol. Cell Proteom. 15, 755–764 (2016).

    Article  Google Scholar 

  93. Lv, X. et al. A positive role for polycomb in transcriptional regulation via H4K20me1. Cell Res. 27, 594 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. 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).

    Article  CAS  PubMed  Google Scholar 

  95. Zhu, Y. et al. Predicting enhancer transcription and activity from chromatin modifications. Nucleic Acids Res. 41, 10032–10043 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Rinzema, N. J. et al. Building regulatory landscapes reveals that an enhancer can recruit cohesin to create contact domains, engage CTCF sites and activate distant genes. Nat. Struct. Mol. Biol. 29, 563–574 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  99. Andrey, G. et al. Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding. Genome Res. 27, 223–233 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013).

    Article  CAS  PubMed  Google Scholar 

  101. Whyte, W. A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Pott, S. & Lieb, J. D. What are super-enhancers? Nat. Genet. 47, 8–12 (2015).

    Article  CAS  PubMed  Google Scholar 

  103. Grosveld, F., van Staalduinen, J. & Stadhouders, R. Transcriptional regulation by (super)enhancers: from discovery to mechanisms. Annu. Rev. Genom. Hum. Genet. 22, 127–146 (2021).

    Article  Google Scholar 

  104. Zhang, Y. et al. Super-silencers regulated by chromatin interactions control apoptotic genes. bioRxiv https://doi.org/10.1101/2022.01.17.476559 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  105. Cannavò, E. et al. Shadow enhancers are pervasive features of developmental regulatory networks. Curr. Biol. 26, 38–51 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  106. Osterwalder, M. et al. Enhancer redundancy provides phenotypic robustness in mammalian development. Nature 554, 239–243 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Kvon, E. Z., Waymack, R., Gad, M. & Wunderlich, Z. Enhancer redundancy in development and disease. Nat. Rev. Genet. 22, 324–336 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Huang, Z. et al. The corepressors GPS2 and SMRT control enhancer and silencer remodeling via eRNA transcription during inflammatory activation of macrophages. Mol. Cell 81, 953–968 e959 (2021).

    Article  CAS  PubMed  Google Scholar 

  109. Huang, D. & Ovcharenko, I. Enhancer-silencer transitions in the human genome. Genome Res. 32, 437–448 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  110. Erceg, J. et al. Dual functionality of cis-regulatory elements as developmental enhancers and Polycomb response elements. Genes Dev. 31, 590–602 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Peng, G. H., Ahmad, O., Ahmad, F., Liu, J. & Chen, S. The photoreceptor-specific nuclear receptor Nr2e3 interacts with Crx and exerts opposing effects on the transcription of rod versus cone genes. Hum. Mol. Genet. 14, 747–764 (2005).

    Article  CAS  PubMed  Google Scholar 

  112. Stampfel, G. et al. Transcriptional regulators form diverse groups with context-dependent regulatory functions. Nature 528, 147–151 (2015).

    Article  CAS  PubMed  Google Scholar 

  113. White, M. A., Myers, C. A., Corbo, J. C. & Cohen, B. A. Massively parallel in vivo enhancer assay reveals that highly local features determine the cis-regulatory function of ChIP-seq peaks. Proc. Natl Acad. Sci. USA 110, 11952–11957 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Lex, R. K. et al. GLI transcriptional repression regulates tissue-specific enhancer activity in response to Hedgehog signaling. eLife 9, e50670 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Cohen, M., Page, K. M., Perez-Carrasco, R., Barnes, C. P. & Briscoe, J. A theoretical framework for the regulation of Shh morphogen-controlled gene expression. Development 141, 3868–3878 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Halfon, M. S. Silencers, enhancers, and the multifunctional regulatory genome. Trends Genet. 36, 149–151 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Sabaris, G., Laiker, I., Preger-Ben Noon, E. & Frankel, N. Actors with multiple roles: pleiotropic enhancers and the paradigm of enhancer modularity. Trends Genet. 35, 423–433 (2019).

    Article  CAS  PubMed  Google Scholar 

  118. Singh, D. & Yi, S. V. Enhancer pleiotropy, gene expression, and the architecture of human enhancer–gene interactions. Mol. Biol. Evol. 38, 3898–3909 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Uffelmann, E. et al. Genome-wide association studies. Nat. Rev. Methods Prim. 1, 59 (2021).

    Article  CAS  Google Scholar 

  120. Cano-Gamez, E. & Trynka, G. From GWAS to function: using functional genomics to identify the mechanisms underlying complex diseases. Front. Genet. 11, 424 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Edwards, S. L., Beesley, J., French, J. D. & Dunning, A. M. Beyond GWASs: illuminating the dark road from association to function. Am. J. Hum. Genet. 93, 779–797 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. Meyer, K. B. et al. Fine-scale mapping of the FGFR2 breast cancer risk locus: putative functional variants differentially bind FOXA1 and E2F1. Am. J. Hum. Genet. 93, 1046–1060 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Campbell, T. M. et al. FGFR2 risk SNPs confer breast cancer risk by augmenting oestrogen responsiveness. Carcinogenesis 37, 741–750 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Zheng, W. et al. Genome-wide association study identifies a new breast cancer susceptibility locus at 6q25.1. Nat. Genet. 41, 324–328 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Turnbull, C. et al. Genome-wide association study identifies five new breast cancer susceptibility loci. Nat. Genet. 42, 504–507 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Dunning, A. M. et al. Breast cancer risk variants at 6q25 display different phenotype associations and regulate ESR1, RMND1 and CCDC170. Nat. Genet. 48, 374–386 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  128. High, F. A. & Epstein, J. A. The multifaceted role of Notch in cardiac development and disease. Nat. Rev. Genet. 9, 49–61 (2008).

    Article  CAS  PubMed  Google Scholar 

  129. Wang, Y. et al. Family-based whole-genome sequencing identifies compound heterozygous protein-coding and noncoding mutations in tetralogy of Fallot. Gene 741, 144555 (2020).

    Article  CAS  PubMed  Google Scholar 

  130. Mika, K. M. & Lynch, V. J. An ancient fecundability-associated polymorphism switches a repressor into an enhancer of endometrial TAP2 expression. Am. J. Hum. Genet. 99, 1059–1071 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Kapoor, A. et al. Multiple SCN5A variant enhancers modulate its cardiac gene expression and the QT interval. Proc. Natl Acad. Sci. USA 116, 10636–10645 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Kaukonen, M. et al. A putative silencer variant in a spontaneous canine model of retinitis pigmentosa. PLoS Genet. 16, e1008659 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. Deplancke, B., Alpern, D. & Gardeux, V. The genetics of transcription factor DNA binding variation. Cell 166, 538–554 (2016).

    Article  CAS  PubMed  Google Scholar 

  134. Carrasco Pro, S., Bulekova, K., Gregor, B., Labadorf, A. & Fuxman Bass, J. I. Prediction of genome-wide effects of single nucleotide variants on transcription factor binding. Sci. Rep. 10, 17632 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. Gan, K. A., Carrasco Pro, S., Sewell, J. A. & Fuxman Bass, J. I. Identification of single nucleotide non-coding driver mutations in cancer. Front. Genet. 9, 16 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  136. Wang, Y. et al. SNP rs17079281 decreases lung cancer risk through creating an YY1-binding site to suppress DCBLD1 expression. Oncogene 39, 4092–4102 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  137. Boltsis, I., Grosveld, F., Giraud, G. & Kolovos, P. Chromatin conformation in development and disease. Front. Cell Dev. Biol. 9, 723859 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  138. Osman, N., Shawky, A.-E.-M. & Brylinski, M. Exploring the effects of genetic variation on gene regulation in cancer in the context of 3D genome structure. BMC Genom. Data 23, 13 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. Lu, L. et al. Robust Hi-C maps of enhancer-promoter interactions reveal the function of non-coding genome in neural development and diseases. Mol. Cell 79, 521–534.e515 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  140. Liu, S. et al. Systematic identification of regulatory variants associated with cancer risk. Genome Biol. 18, 194 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  141. Ouwerkerk, A. F. V. et al. Identification of functional variant enhancers associated with atrial fibrillation. Cir. Res. 127, 229–243 (2020).

    Article  Google Scholar 

  142. van Arensbergen, J. et al. High-throughput identification of human SNPs affecting regulatory element activity. Nat. Genet. 51, 1160–1169 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  143. Uebbing, S. et al. Massively parallel discovery of human-specific substitutions that alter enhancer activity. Proc. Natl Acad. Sci. USA 118, e2007049118 (2021).

    Article  CAS  PubMed  Google Scholar 

  144. Platt, O. S. et al. Pain in sickle cell disease: rates and risk factors. N. Engl. J. Med. 325, 11–16 (1991).

    Article  CAS  PubMed  Google Scholar 

  145. Platt, O. S. et al. Mortality in sickle cell disease-life expectancy and risk factors for early death. N. Engl. J. Med. 330, 1639–1644 (1994).

    Article  CAS  PubMed  Google Scholar 

  146. Steinberg, M. H. Fetal hemoglobin in sickle cell anemia. Blood 136, 2392–2400 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  147. Sankaran, V. G. et al. Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science 322, 1839–1842 (2008).

    Article  CAS  PubMed  Google Scholar 

  148. Bauer, D. E. et al. An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level. Science 342, 253–257 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. Bauer, D. E. & Orkin, S. H. Hemoglobin switching’s surprise: the versatile transcription factor BCL11A is a master repressor of fetal hemoglobin. Curr. Opin. Genet. Dev. 33, 62–70 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  150. Demirci, S., Leonard, A., Essawi, K. & Tisdale, J. F. CRISPR-Cas9 to induce fetal hemoglobin for the treatment of sickle cell disease. Mol. Ther. Methods Clin. Dev. 23, 276–285 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  151. Chang, K.-H. et al. Long-term engraftment and fetal globin induction upon BCL11A gene editing in bone-marrow-derived CD34+ hematopoietic stem and progenitor cells. Mol. Ther. Methods Clin. Dev. 4, 137–148 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  153. Wu, Y. et al. Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat. Med. 25, 776–783 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  154. Demirci, S. et al. BCL11A enhancer-edited hematopoietic stem cells persist in rhesus monkeys without toxicity. J. Clin. Invest. 130, 6677–6687 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  155. Liu, N. et al. Direct promoter repression by BCL11A controls the fetal to adult hemoglobin switch. Cell 173, 430–442.e417 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  156. Frangoul, H. et al. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N. Engl. J. Med. 384, 252–260 (2020).

    Article  PubMed  Google Scholar 

  157. Chang, K. H. et al. Long-term engraftment and fetal globin induction upon BCL11A gene editing in bone-marrow-derived CD34+ hematopoietic stem and progenitor cells. Mol. Ther. Methods Clin. Dev. 4, 137–148 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  158. Zeng, W. et al. SilencerDB: a comprehensive database of silencers. Nucleic Acids Res. 49, D221–D228 (2021).

    Article  CAS  PubMed  Google Scholar 

  159. Hoffman, M. M. et al. Integrative annotation of chromatin elements from ENCODE data. Nucleic Acids Res. 41, 827–841 (2013).

    Article  CAS  PubMed  Google Scholar 

  160. Moore, J. E. et al. Expanded encyclopaedias of DNA elements in the human and mouse genomes. Nature 583, 699–710 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  161. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  163. Gasperini, M. et al. A genome-wide framework for mapping gene regulation via cellular genetic screens. Cell 176, 377–390.e319 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  164. Inoue, F. & Ahituv, N. Decoding enhancers using massively parallel reporter assays. Genomics 106, 159–164 (2015).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  166. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by a KWF Young Investigator Grant from the Dutch Cancer Society (11707) and an ERC Starting Grant from the European Research Council (950655-Silencer) awarded to B.P.

Author information

Authors and Affiliations

Authors

Contributions

B.P., J.H.v.W. and F.L.H. researched data for the article, wrote the article and reviewed and/or edited the manuscript before submission. M.P.S. contributed substantially to discussion of the content.

Corresponding authors

Correspondence to Baoxu Pang or Michael P. Snyder.

Ethics declarations

Competing interests

M.P.S. is a founder and member of the science advisory boards of Personalis, SensOmics, Qbio, January, Mirvie and Filtricine, and a member of the science advisory boards of Genapsys and Epinomics. B.P. and M.P.S. hold a patent on the ReSE screening system and method (WO-2021155369-A1).

Peer review

Peer review information

Nature Reviews Molecular Cell Biology thanks Emily Wong and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

ENCODE: https://www.encodeproject.org/

EnhancerAtlas: http://www.enhanceratlas.org/

SilencerDB: http://health.tsinghua.edu.cn/silencerdb/

VISTA Enhancer Browser: https://enhancer.lbl.gov/

Glossary

Assay for transposase-accessible chromatin using sequencing

(ATAC-seq). A high-throughput technique to identify regions of accessible chromatin genome-wide.

Cardiac QT interval

An electrocardiographic measure of myocardial repolarization.

Chromatin interaction analysis with paired-end tag sequencing

(ChIA-PET). A high-throughput technique to identify genome-wide chromatin interactions mediated by specific factors.

Chromatin looping factors CTCF and cohesion

Architectural proteins that mediate longe-range genomic interactions by contributing to three-dimensional genome organization.

Expression quantitative trait locus

A genomic locus or single-nucleotide polymorphism that is associated with differential gene expression, thereby linking variations in gene expression levels to different genotypes.

Formaldehyde-assisted isolation of regulatory elements

(FAIRE). A high-throughput technique to discover nucleosome-depleted genomic regions, which are indicative of regulatory activity.

Genome-wide association studies

(GWAS). Studies used to identify genomic variants that are statistically associated with a particular trait or disease risk.

Genomic position effects

The influence of the endogenous chromosomal environment on the activity of a gene or a regulatory element.

Hi-C

A chromosome conformation capture technique that maps the three-dimensional organization of the genome.

Insulators

Non-coding regulatory elements with enhancer blocking or barrier function.

Massively parallel reporter assay

(MPRA). A high-throughput technique to simultaneously measure the transcriptional activity of thousands of candidate non-coding regulatory elements.

Self-transcribing active regulatory region sequencing

(STARR-seq). A type of massively parallel reporter assay in which candidate non-coding regulatory elements are cloned downstream of a reporter gene so that their enhancer or silencer activity is reflected in the abundance of the non-coding regulatory element sequence within the pool of plasmid-derived RNA.

‘Shadow’ enhancers

Two or more enhancers with seemingly redundant functions that regulate the same target gene or genes.

Topologically associating domain

Chromatin domain of typically ~100 kb to ~1 Mb, characterized by high intradomain contacts that contribute to NCRE-promoter interactions.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Pang, B., van Weerd, J.H., Hamoen, F.L. et al. Identification of non-coding silencer elements and their regulation of gene expression. Nat Rev Mol Cell Biol (2022). https://doi.org/10.1038/s41580-022-00549-9

Download citation

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41580-022-00549-9

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing