Gene Regulatory Elements

  • Nadav AhituvEmail author


While the annotation and functional characterization of the 2% of our genome that encodes for protein has been extremely successful, the remaining 98% still remains primarily uncharted territory. Within this territory reside gene regulatory sequences that instruct genes when, where, and at what levels to turn on or off. There is abundant evidence, as described in this book, that nucleotide and epigenetic changes in these gene regulatory sequences can lead to human disease. In this chapter, we will define the different types of gene regulatory elements (promoters, enhancers, silencers, and insulators) and how to identify and functionally characterize them.


Promoter Enhancer Silencer Insulator Locus control region Transcription factors Nucleosome positioning DNase I hypersensitive sites ChIP 3C 



TFIIB recognition element


Initiator element


Downstream core promoter element


TATA-binding protein


Transcription factor IID


Transcription factor IIB


Transcription start site


Transcription factor binding sites


Sonic hedgehog


Nipped-B homolog


CCCTC-binding factor


Locus control region


DNase I hypersensitive sites


Chromatin immunoprecipitation


Chromatin conformation capture


Yeast artificial chromosome


Bacterial artificial chromosome


  1. Ahituv N, Zhu Y et al (2007) Deletion of ultraconserved elements yields viable mice. PLoS Biol 5(9):e234PubMedCrossRefGoogle Scholar
  2. Ameres SL, Drueppel L et al (2005) Inducible DNA-loop formation blocks transcriptional activation by an SV40 enhancer. EMBO J 24(2):358–367PubMedCrossRefGoogle Scholar
  3. Barski A, Cuddapah S et al (2007) High-resolution profiling of histone methylations in the human genome. Cell 129(4):823–837PubMedCrossRefGoogle Scholar
  4. Bernstein BE, Kamal M et al (2005) Genomic maps and comparative analysis of histone modifications in human and mouse. Cell 120(2):169–181PubMedCrossRefGoogle Scholar
  5. Bi X, Yu Q et al (2004) Formation of boundaries of transcriptionally silent chromatin by nucleosome-excluding structures. Mol Cell Biol 24(5):2118–2131PubMedCrossRefGoogle Scholar
  6. Birney E, Stamatoyannopoulos JA et al (2007) Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447(7146):799–816PubMedCrossRefGoogle Scholar
  7. Blanton J, Gaszner M et al (2003) Protein:protein interactions and the pairing of boundary elements in vivo. Genes Dev 17(5):664–675PubMedCrossRefGoogle Scholar
  8. Boffelli D, Nobrega MA et al (2004) Comparative genomics at the vertebrate extremes. Nat Rev Genet 5(6):456–465PubMedCrossRefGoogle Scholar
  9. Bulger M, Groudine M (2011) Functional and mechanistic diversity of distal transcription enhancers. Cell 144(3):327–339PubMedCrossRefGoogle Scholar
  10. Burke TW, Kadonaga JT (1997) The downstream core promoter element, DPE, is conserved from Drosophila to humans and is recognized by TAFII60 of Drosophila. Genes Dev 11(22):3020–3031PubMedCrossRefGoogle Scholar
  11. Bushey AM, Dorman ER et al (2008) Chromatin insulators: regulatory mechanisms and epigenetic inheritance. Mol Cell 32(1):1–9PubMedCrossRefGoogle Scholar
  12. Butler JE, Kadonaga JT (2002) The RNA polymerase II core promoter: a key component in the regulation of gene expression. Genes Dev 16(20):2583–2592PubMedCrossRefGoogle Scholar
  13. Byrd K, Corces VG (2003) Visualization of chromatin domains created by the gypsy insulator of Drosophila. J Cell Biol 162(4):565–574PubMedCrossRefGoogle Scholar
  14. Calhoun VC, Stathopoulos A et al (2002) Promoter-proximal tethering elements regulate enhancer-promoter specificity in the Drosophila Antennapedia complex. Proc Natl Acad Sci USA 99(14):9243–9247PubMedCrossRefGoogle Scholar
  15. Carroll SB (2005) Evolution at two levels: on genes and form. PLoS Biol 3(7):e245PubMedCrossRefGoogle Scholar
  16. Clamp M, Fry B et al (2007) Distinguishing protein-coding and noncoding genes in the human genome. Proc Natl Acad Sci USA 104(49):19428–19433PubMedCrossRefGoogle Scholar
  17. Crawford GE, Davis S et al (2006) DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays. Nat Methods 3(7):503–509PubMedCrossRefGoogle Scholar
  18. Cretekos CJ, Wang Y et al (2008) Regulatory divergence modifies limb length between mammals. Genes Dev 22(2):141–151PubMedCrossRefGoogle Scholar
  19. Creyghton MP, Cheng AW et al (2010) Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc Natl Acad Sci USA 107(50):21931–21936PubMedCrossRefGoogle Scholar
  20. Dermitzakis ET, Reymond A et al (2005) Conserved non-genic sequences – an unexpected feature of mammalian genomes. Nat Rev Genet 6(2):151–157PubMedCrossRefGoogle Scholar
  21. Dorsett D (2011) Cohesin: genomic insights into controlling gene transcription and development. Curr Opin Genet Dev 21(2):199–206PubMedCrossRefGoogle Scholar
  22. Ernst J, Kheradpour P et al (2011) Mapping and analysis of chromatin state dynamics in nine human cell types. Nature 473(7345):43–49PubMedCrossRefGoogle Scholar
  23. Filippova GN (2008) Genetics and epigenetics of the multifunctional protein CTCF. Curr Top Dev Biol 80:337–360PubMedCrossRefGoogle Scholar
  24. Fisher S, Grice EA et al (2006) Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish. Nat Protoc 1(3):1297–1305PubMedCrossRefGoogle Scholar
  25. Fullwood MJ, Liu MH et al (2009) An oestrogen-receptor-alpha-bound human chromatin interactome. Nature 462(7269):58–64PubMedCrossRefGoogle Scholar
  26. Gardiner-Garden M, Frommer M (1987) CpG islands in vertebrate genomes. J Mol Biol 196(2):261–282PubMedCrossRefGoogle Scholar
  27. Gaszner M, Felsenfeld G (2006) Insulators: exploiting transcriptional and epigenetic mechanisms. Nat Rev Genet 7(9):703–713PubMedCrossRefGoogle Scholar
  28. Geyer PK (1997) The role of insulator elements in defining domains of gene expression. Curr Opin Genet Dev 7(2):242–248PubMedCrossRefGoogle Scholar
  29. Guelen L, Pagie L et al (2008) Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature 453(7197):948–951PubMedCrossRefGoogle Scholar
  30. Guenther MG, Levine SS et al (2007) A chromatin landmark and transcription initiation at most promoters in human cells. Cell 130(1):77–88PubMedCrossRefGoogle Scholar
  31. Harris MB, Mostecki J et al (2005) Repression of an interleukin-4-responsive promoter requires cooperative BCL-6 function. J Biol Chem 280(13):13114–13121PubMedCrossRefGoogle Scholar
  32. Heintzman ND, Stuart RK et al (2007) Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet 39(3):311–318PubMedCrossRefGoogle Scholar
  33. Heintzman ND, Hon GC et al (2009) Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459(7243):108–112PubMedCrossRefGoogle Scholar
  34. Hobert O (2010) Gene regulation: enhancers stepping out of the shadow. Curr Biol 20(17):R697–R699PubMedCrossRefGoogle Scholar
  35. Hong JW, Hendrix DA et al (2008) Shadow enhancers as a source of evolutionary novelty. Science 321(5894):1314PubMedCrossRefGoogle Scholar
  36. Johnson DS, Mortazavi A et al (2007) Genome-wide mapping of in vivo protein-DNA interactions. Science 316(5830):1497–1502PubMedCrossRefGoogle Scholar
  37. Kagey MH, Newman JJ et al (2010) Mediator and cohesin connect gene expression and chromatin architecture. Nature 467(7314):430–435PubMedCrossRefGoogle Scholar
  38. Khokha MK, Loots GG (2005) Strategies for characterising cis-regulatory elements in Xenopus. Brief Funct Genomic Proteom 4(1):58–68CrossRefGoogle Scholar
  39. Kim TH, Abdullaev ZK et al (2007) Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome. Cell 128(6):1231–1245PubMedCrossRefGoogle Scholar
  40. Kim TK, Hemberg M et al (2010) Widespread transcription at neuronal activity-regulated enhancers. Nature 465(7295):182–187PubMedCrossRefGoogle Scholar
  41. Kong S, Bohl D et al (1997) Transcription of the HS2 enhancer toward a cis-linked gene is independent of the orientation, position, and distance of the enhancer relative to the gene. Mol Cell Biol 17(7):3955–3965PubMedGoogle Scholar
  42. Korzh V (2007) Transposons as tools for enhancer trap screens in vertebrates. Genome Biol 8(Suppl 1):S8PubMedCrossRefGoogle Scholar
  43. Kutach AK, Kadonaga JT (2000) The downstream promoter element DPE appears to be as widely used as the TATA box in Drosophila core promoters. Mol Cell Biol 20(13):4754–4764PubMedCrossRefGoogle Scholar
  44. Lagrange T, Kapanidis AN et al (1998) New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB. Genes Dev 12(1):34–44PubMedCrossRefGoogle Scholar
  45. Lanzuolo C, Roure V et al (2007) Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex. Nat Cell Biol 9(10):1167–1174PubMedCrossRefGoogle Scholar
  46. Lettice LA, Heaney SJ et al (2003) A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum Mol Genet 12(14):1725–1735PubMedCrossRefGoogle Scholar
  47. Li L, He S et al (2004) Gene regulation by Sp1 and Sp3. Biochem Cell Biol 82(4):460–471PubMedCrossRefGoogle Scholar
  48. Lieberman-Aiden E, van Berkum NL et al (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326(5950):289–293PubMedCrossRefGoogle Scholar
  49. Ling J, Ainol L et al (2004) HS2 enhancer function is blocked by a transcriptional terminator inserted between the enhancer and the promoter. J Biol Chem 279(49):51704–51713PubMedCrossRefGoogle Scholar
  50. Lomvardas S, Barnea G et al (2006) Interchromosomal interactions and olfactory receptor choice. Cell 126(2):403–413PubMedCrossRefGoogle Scholar
  51. Malik S, Roeder RG (2010) The metazoan mediator co-activator complex as an integrative hub for transcriptional regulation. Nat Rev Genet 11(11):761–772PubMedCrossRefGoogle Scholar
  52. McLean CY, Reno PL et al (2011) Human-specific loss of regulatory DNA and the evolution of human-specific traits. Nature 471(7337):216–219PubMedCrossRefGoogle Scholar
  53. Mikkelsen TS, Ku M et al (2007) Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448(7153):553–560PubMedCrossRefGoogle Scholar
  54. Mortlock DP, Guenther C et al (2003) A general approach for identifying distant regulatory elements applied to the Gdf6 gene. Genome Res 13(9):2069–2081PubMedCrossRefGoogle Scholar
  55. Nagy A, Gertsenstein M et al (2002) Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor, New YorkGoogle Scholar
  56. Neznanov N, Umezawa A et al (1997) A regulatory element within a coding exon modulates keratin 18 gene expression in transgenic mice. J Biol Chem 272(44):27549–27557PubMedCrossRefGoogle Scholar
  57. Oki M, Valenzuela L et al (2004) Barrier proteins remodel and modify chromatin to restrict silenced domains. Mol Cell Biol 24(5):1956–1967PubMedCrossRefGoogle Scholar
  58. Ong CT, Corces VG (2011) Enhancer function: new insights into the regulation of tissue-specific gene expression. Nat Rev Genet 12(4):283–293PubMedCrossRefGoogle Scholar
  59. Parelho V, Hadjur S et al (2008) Cohesins functionally associate with CTCF on mammalian chromosome arms. Cell 132(3):422–433PubMedCrossRefGoogle Scholar
  60. Parinov S, Kondrichin I et al (2004) Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo. Dev Dyn 231(2):449–459PubMedCrossRefGoogle Scholar
  61. Pennacchio LA, Ahituv N et al (2006) In vivo enhancer analysis of human conserved non-coding sequences. Nature 444(7118):499–502PubMedCrossRefGoogle Scholar
  62. Petrykowska HM, Vockley CM et al (2008) Detection and characterization of silencers and enhancer-blockers in the greater CFTR locus. Genome Res 18(8):1238–1246PubMedCrossRefGoogle Scholar
  63. Prabhakar S, Visel A et al (2008) Human-specific gain of function in a developmental enhancer. Science 321(5894):1346–1350PubMedCrossRefGoogle Scholar
  64. Privalsky ML (2004) The role of corepressors in transcriptional regulation by nuclear hormone receptors. Annu Rev Physiol 66:315–360PubMedCrossRefGoogle Scholar
  65. Rada-Iglesias A, Bajpai R et al (2011) A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470(7333):279–283PubMedCrossRefGoogle Scholar
  66. Recillas-Targa F, Pikaart MJ et al (2002) Position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities. Proc Natl Acad Sci USA 99(10):6883–6888PubMedCrossRefGoogle Scholar
  67. Riethoven JJ (2010) Regulatory regions in DNA: promoters, enhancers, silencers, and insulators. Methods 674:33–42Google Scholar
  68. Sankaran VG, Xu J et al (2010) Advances in the understanding of haemoglobin switching. Br J Haematol 149(2):181–194PubMedCrossRefGoogle Scholar
  69. Smale ST, Kadonaga JT (2003) The RNA polymerase II core promoter. Annu Rev Biochem 72:449–479PubMedCrossRefGoogle Scholar
  70. Song L, Crawford GE (2010) DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells. Cold 2010(2):pdb.prot5384Google Scholar
  71. Srinivasan L, Atchison ML (2004) YY1 DNA binding and PcG recruitment requires CtBP. Genes Dev 18(21):2596–2601PubMedCrossRefGoogle Scholar
  72. Suzuki Y, Tsunoda T et al (2001) Identification and characterization of the potential promoter regions of 1031 kinds of human genes. Genome Res 11(5):677–684PubMedCrossRefGoogle Scholar
  73. Thomas JW, Touchman JW et al (2003) Comparative analyses of multi-species sequences from targeted genomic regions. Nature 424(6950):788–793PubMedCrossRefGoogle Scholar
  74. Tiwari VK, McGarvey KM et al (2008) PcG proteins, DNA methylation, and gene repression by chromatin looping. PLoS Biol 6(12):2911–2927PubMedCrossRefGoogle Scholar
  75. Tumpel S, Cambronero F et al (2008) A regulatory module embedded in the coding region of Hoxa2 controls expression in rhombomere 2. Proc Natl Acad Sci USA 105(51):20077–20082, Epub 2008 Dec 22PubMedCrossRefGoogle Scholar
  76. Uchikawa M (2008) Enhancer analysis by chicken embryo electroporation with aid of genome comparison. Dev Growth Differ 50(6):467–474PubMedCrossRefGoogle Scholar
  77. van Berkum NL, Dekker J (2009) Determining spatial chromatin organization of large genomic regions using 5C technology. Methods Mol Biol 567:189–213PubMedCrossRefGoogle Scholar
  78. Vassetzky Y, Gavrilov A et al (2009) Chromosome conformation capture (from 3C to 5C) and its ChIP-based modification. Methods Mol Biol 567:171–188PubMedCrossRefGoogle Scholar
  79. Visel A, Prabhakar S et al (2008) Ultraconservation identifies a small subset of extremely constrained developmental enhancers. Nat Genet 6:6Google Scholar
  80. Visel A, Akiyama JA et al (2009a) Functional autonomy of distant-acting human enhancers. Genomics 93(6):509–513PubMedCrossRefGoogle Scholar
  81. Visel A, Blow MJ et al (2009b) ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457(7231):854–858PubMedCrossRefGoogle Scholar
  82. Weintraub H, Groudine M (1976) Chromosomal subunits in active genes have an altered conformation. Science 193(4256):848–856PubMedCrossRefGoogle Scholar
  83. Wendt KS, Yoshida K et al (2008) Cohesin mediates transcriptional insulation by CCCTC-binding factor. Nature 451(7180):796–801PubMedCrossRefGoogle Scholar
  84. Wood AJ, Severson AF et al (2010) Condensin and cohesin complexity: the expanding repertoire of functions. Nat Rev Genet 11(6):391–404PubMedCrossRefGoogle Scholar
  85. Woolfe A, Goodson M et al (2005) Highly conserved non-coding sequences are associated with vertebrate development. PLoS Biol 3(1):e7PubMedCrossRefGoogle Scholar
  86. Yusufzai TM, Tagami H et al (2004) CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species. Mol Cell 13(2):291–298PubMedCrossRefGoogle Scholar
  87. Zhao H, Dean A (2004) An insulator blocks spreading of histone acetylation and interferes with RNA polymerase II transfer between an enhancer and gene. Nucleic Acids Res 32(16): 4903–4919, Print 2004PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Bioengineering and Therapeutic Sciences, Institute for Human GeneticsUniversity of California San FranciscoSan FranciscoUSA

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