Methods of massive parallel reporter assays for investigation of enhancers

The correct deployment of genetic programs for development and differentiation relies on finely coordinated regulation of specific gene sets. Genomic regulatory elements play an exceptional role in this process. There are few types of gene regulatory elements, including promoters, enhancers, insulators and silencers. Alterations of gene regulatory elements may cause various pathologies, including cancer, congenital disorders and autoimmune diseases. The development of high-throughput genomic assays has made it possible to significantly accelerate the accumulation of information about the characteristic epigenetic properties of regulatory elements. In combination with high-throughput studies focused on the genome-wide distribution of epigenetic marks, regulatory proteins and the spatial structure of chromatin, this significantly expands the understanding of the principles of epigenetic regulation of genes and allows potential regulatory elements to be searched for in silico. However, common experimental approaches used to study the local characteristics of chromatin have a number of technical limitations that may reduce the reliability of computational identification of genomic regulatory sequences. Taking into account the variability of the functions of epigenetic determinants and complex multicomponent regulation of genomic elements activity, their functional verification is often required. A plethora of methods have been developed to study the functional role of regulatory elements on the genome scale. Common experimental approaches for in silico identification of regulatory elements and their inherent technical limitations will be described. The present review is focused on original high-throughput methods of enhancer activity reporter analysis that are currently used to validate predicted regulatory elements and to perform de novo searches. The methods described allow assessing the functional role of the nucleotide sequence of a regulatory element, to determine its exact boundaries and to assess the influence of the local state of chromatin on the activity of enhancers and gene expression. These approaches have contributed substantially to the understanding of the fundamental principles of gene regulation.

1. Akhtar-Zaidi B., Cowper-Sal-lari R., Corradin O., Saiakhova A., Bartels C.F., Balasubramanian D., Myeroff L., Lutterbaugh J., Jarrar A., Kalady M.F., Willis J., Moore J.H., Tesar P.J., Laframboise T., Markowitz S., Lupien M., Scacheri P.C. Epigenomic enhancer prof iling defines a signature of colon cancer. Science. 2012;336:736-739.

2. Akhtar W., de Jong J., Pindyurin A.V., Pagie L., Meuleman W., de Ridder J., Berns A., Wessels L.F., van Lohuizen M., van Steensel B. Chromatin position effects assayed by thousands of reporters integrated in parallel. Cell. 2013;154:914-927.

3. Andersson R., Gebhard C., Miguel-Escalada I., Hoof I., Bornholdt J., Boyd M., Chen Y., …, Suzuki H., Hayashizaki Y., Muller F., Forrest A.R.R., Carninci P., Rehli M., Sandelin A. An atlas of active enhancers across human cell types and tissues. Nature. 2014;507: 455-461.

4. Andersson R., Sandelin A. Determinants of enhancer and promoter activities of regulatory elements. Nat. Rev. Genet. 2020;21:71-87.

5. Arnold C.D., Gerlach D., Stelzer C., Boryn L.M., Rath M., Stark A. Genome-wide quantitative enhancer activity maps identified by STARR-seq. Science. 2013;339:1074-1077.

6. Banerji J., Rusconi S., Schaffner W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell. 1981;27:299- 308.

7. Barakat T.S., Halbritter F., Zhang M., Rendeiro A.F., Perenthaler E., Bock C., Chambers I. Functional dissection of the enhancer repertoire in human embryonic stem cells. Cell Stem Cell. 2018;23:276- 288 e278.

8. Barski A., Cuddapah S., Cui K., Roh T.Y., Schones D.E., Wang Z., Wei G., Chepelev I., Zhao K. High-resolution profiling of histone methylations in the human genome. Cell. 2007;129:823-837.

9. Bernstein B.E., Kamal M., Lindblad-Toh K., Bekiranov S., Bailey D.K., Huebert D.J., McMahon S., Karlsson E.K., Kulbokas E.J. 3rd, Gingeras T.R., Schreiber S.L., Lander E.S. Genomic maps and comparative analysis of histone modifications in human and mouse. Cell. 2005;120:169-181.

10. Bernstein B.E., Stamatoyannopoulos J.A., Costello J.F., Ren B., Milosavljevic A., Meissner A., Kellis M., Marra M.A., Beaudet A.L., Ecker J.R., Farnham P.J., Hirst M., Lander E.S., Mikkelsen T.S.,

11. Thomson J.A. The NIH roadmap epigenomics mapping consortium. Nat. Biotechnol. 2010;28:1045-1048. Birney E., Stamatoyannopoulos J.A., Dutta A., Guigo R., Gingeras T.R., Margulies E.H., Weng Z., …, Lander E.S., Koriabine M., Nefedov M., Osoegawa K., Yoshinaga Y., Zhu B., de Jong P.J. Identification and analysis of functional elements in 1 % of the human genome by the ENCODE pilot project. Nature. 2007;447: 799-816.

12. Bonn S., Zinzen R.P., Girardot C., Gustafson E.H., Perez-Gonzalez A., Delhomme N., Ghavi-Helm Y., Wilczynski B., Riddell A., Furlong E.E. Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development. Nat. Genet. 2012;44:148-156.

13. Bradner J.E., Hnisz D., Young R.A. Transcriptional addiction in cancer. Cell. 2017;168:629-643.

14. Buenrostro J.D., Giresi P.G., Zaba L.C., Chang H.Y., Greenleaf W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods. 2013;10:1213-1218.

15. Cai H., Levine M. Modulation of enhancer-promoter interactions by insulators in the Drosophila embryo. Nature. 1995;376:533-536.

16. Canver M.C., Bauer D.E., Orkin S.H. Functional interrogation of noncoding DNA through CRISPR genome editing. Methods. 2017; (121-122):118-129.

17. Chatterjee S., Ahituv N. Gene regulatory elements, major drivers of human disease. Annu. Rev. Genomics Hum. Genet. 2017;18:45-63.

18. Chavez A., Scheiman J., Vora S., Pruitt B.W., Tuttle M., Eswar P.R.I., Lin S., Kiani S., Guzman C.D., Wiegand D.J., Ter-Ovanesyan D., Braff J.L., Davidsohn N., Housden B.E., Perrimon N., Weiss R., Aach J., Collins J.J., Church G.M. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods. 2015;12:326-328.

19. Corradin O., Saiakhova A., Akhtar-Zaidi B., Myeroff L., Willis J., Cowper-Sallari R., Lupien M., Markowitz S., Scacheri P.C. Combinatorial effects of multiple enhancer variants in linkage disequilibrium dictate levels of gene expression to confer susceptibility to common traits. Genome Res. 2014;24:1-13.

20. Creyghton M.P., Cheng A.W., Welstead G.G., Kooistra T., Carey B.W., Steine E.J., Hanna J., Lodato M.A., Frampton G.M., Sharp P.A., Boyer L.A., Young R.A., Jaenisch R. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. USA. 2010;107:21931-21936.

21. Crocker J., Abe N., Rinaldi L., McGregor A.P., Frankel N., Wang S., Alsawadi A., Valenti P., Plaza S., Payre F., Mann R.S., Stern D.L. Low affinity binding site clusters confer hox specificity and regulatory robustness. Cell. 2015;160:191-203.

22. Crocker J., Noon E.P., Stern D.L. The soft touch: low-affinity transcription factor binding sites in development and evolution. Curr. Top. Dev. Biol. 2016;117:455-469.

23. Dekker J., Rippe K., Dekker M., Kleckner N. Capturing chromosome conformation. Science. 2002;295:1306-1311.

24. Diao Y., Li B., Meng Z., Jung I., Lee A.Y., Dixon J., Maliskova L., Guan K.L., Shen Y., Ren B. A new class of temporarily phenotypic enhancers identified by CRISPR/Cas9-mediated genetic screening. Genome Res. 2016;26:397-405.

25. Dickel D.E., Ypsilanti A.R., Pla R., Zhu Y., Barozzi I., Mannion B.J., Khin Y.S., Fukuda-Yuzawa Y., Plajzer-Frick I., Pickle C.S., Lee E.A., Harrington A.N., Pham Q.T., Garvin T.H., Kato M., Osterwalder M., Akiyama J.A., Afzal V., Rubenstein J.L.R., Pennacchio L.A., Visel A. Ultraconserved enhancers are required for normal development. Cell. 2018;172:491-499.e415.

26. Dickel D.E., Zhu Y., Nord A.S., Wylie J.N., Akiyama J.A., Afzal V., Plajzer-Frick I., Kirkpatrick A., Gottgens B., Bruneau B.G., Visel A., Pennacchio L.A. Function-based identification of mammalian enhancers using site-specific integration. Nat. Meth. 2014;11:566-571.

27. Dixon J.R., Jung I., Selvaraj S., Shen Y., Antosiewicz-Bourget J.E., Lee A.Y., Ye Z., Kim A., Rajagopal N., Xie W., Diao Y., Liang J., Zhao H., Lobanenkov V.V., Ecker J.R., Thomson J.A., Ren B. Chromatin architecture reorganization during stem cell differentiation. Nature. 2015;518:331-336.

28. Dixon J.R., Selvaraj S., Yue F., Kim A., Li Y., Shen Y., Hu M., Liu J.S., Ren B. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature. 2012;485:376-380.

29. Ernst J., Kheradpour P., Mikkelsen T.S., Shoresh N., Ward L.D., Epstein C.B., Zhang X., Wang L., Issner R., Coyne M., Ku M., Durham T., Kellis M., Bernstein B.E. Mapping and analysis of chromatin state dynamics in nine human cell types. Nature. 2011;473:43-49.

30. Fang R., Yu M., Li G., Chee S., Liu T., Schmitt A.D., Ren B. Mapping of long-range chromatin interactions by proximity ligation-assisted ChIP-seq. Cell Res. 2016;26:1345-1348.

31. Farley E.K., Olson K.M., Zhang W., Brandt A.J., Rokhsar D.S., Levine M.S. Suboptimization of developmental enhancers. Science. 2015;350:325-328.

32. Forrest A.R., Kawaji H., Rehli M., Baillie J.K., de Hoon M.J., Haberle V., Lassmann T., …, Bajic V.B., Taylor M.S., Makeev V.J., Sandelin A., Hume D.A., Carninci P., Hayashizaki Y. A promoter-level mammalian expression atlas. Nature. 2014;507:462-470.

33. Fullwood M.J., Ruan Y. ChIP-based methods for the identification of long-range chromatin interactions. J. Cell. Biochem. 2009;107: 30-39.

34. Furlong E.E.M., Levine M. Developmental enhancers and chromosome topology. Science. 2018;361:1341-1345.

35. Gasperini M., Hill A.J., McFaline-Figueroa J.L., Martin B., Kim S., Zhang M.D., Jackson D., Leith A., Schreiber J., Noble W.S., Trapnell C., Ahituv N., Shendure J. A genome-wide framework for mapping gene regulation via cellular genetic screens. Cell. 2019;176: 1516.

36. Gaulton K.J., Nammo T., Pasquali L., Simon J.M., Giresi P.G., Fogarty M.P., Panhuis T.M., Mieczkowski P., Secchi A., Bosco D., Berney T., Montanya E., Mohlke K.L., Lieb J.D., Ferrer J. A map of open chromatin in human pancreatic islets. Nat. Genet. 2010;42: 255-259.

37. Gavrilov A., Eivazova E., Priozhkova I., Lipinski M., Razin S., Vassetzky Y. Chromosome conformation capture (from 3C to 5C) and its ChIP-based modification. Methods Mol. Biol. 2009;567:171-188.

38. Geyer P.K., Corces V.G. DNA position-specific repression of transcription by a Drosophila zinc finger protein. Genes Dev. 1992;6: 1865-1873.

39. Ghavi-Helm Y., Klein F.A., Pakozdi T., Ciglar L., Noordermeer D., Huber W., Furlong E.E. Enhancer loops appear stable during development and are associated with paused polymerase. Nature. 2014; 512: 96-100.

40. Gomes A.L., Abeel T., Peterson M., Azizi E., Lyubetskaya A., Carvalho L., Galagan J. Decoding ChIP-seq with a double-binding signal refines binding peaks to single-nucleotides and predicts cooperative interaction. Genome Res. 2014;24:1686-1697.

41. Halfon M.S., Carmena A., Gisselbrecht S., Sackerson C.M., Jimenez F., Baylies M.K., Michelson A.M. Ras pathway specificity is determined by the integration of multiple signal-activated and tissuerestricted transcription factors. Cell. 2000;103:63-74.

42. Hammar P., Leroy P., Mahmutovic A., Marklund E.G., Berg O.G., Elf J. The lac repressor displays facilitated diffusion in living cells. Science. 2012;336:1595-1598.

43. Harris M.B., Mostecki J., Rothman P.B. Repression of an interleukin4-responsive promoter requires cooperative BCL-6 function. J. Biol. Chem. 2005;280:13114-13121.

44. Heintzman N.D., Stuart R.K., Hon G., Fu Y., Ching C.W., HawkinsR.D., Barrera L.O., Van Calcar S., Qu C., Ching K.A., Wang W., Weng Z., Green R.D., Crawford G.E., Ren B. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 2007;39:311-318.

45. 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. 2019;29:657- 667.

46. Inoue F., Kircher M., Martin B., Cooper G.M., Witten D.M., McManus M.T., Ahituv N., Shendure J. A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity. Genome Res. 2017;27:38-52.

47. Jung Y.L., Luquette L.J., Ho J.W., Ferrari F., Tolstorukov M., Minoda A., Issner R., Epstein C.B., Karpen G.H., Kuroda M.I., Park P.J. Impact of sequencing depth in ChIP-seq experiments. Nucleic Acids Res. 2014;42:e74.

48. Kaya-Okur H.S., Wu S.J., Codomo C.A., Pledger E.S., Bryson T.D., Henikoff J.G., Ahmad K., Henikoff S. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat. Commun. 2019;10:1930.

49. Kel A.E., Gossling E., Reuter I., Cheremushkin E., Kel-Margoulis O.V., Wingender E. MATCH: A tool for searching transcription factor binding sites in DNA sequences. Nucleic Acids Res. 2003;31:3576- 3579.

50. Kellis M., Wold B., Snyder M.P., Bernstein B.E., Kundaje A., Marinov G.K., Ward L.D., Birney E., Crawford G.E., Dekker J., Dunham I., Elnitski L.L., Farnham P.J., Feingold E.A., Gerstein M., Giddings M.C., Gilbert D.M., Gingeras T.R., Green E.D., Guigo R., Hubbard T., Kent J., Lieb J.D., Myers R.M., Pazin M.J., Ren B., Stamatoyannopoulos J.A., Weng Z., White K.P., Hardison R.C. Defining functional DNA elements in the human genome. Proc. Natl. Acad. Sci. USA. 2014;111:6131-6138.

51. Kellum R., Schedl P. A position-effect assay for boundaries of higher order chromosomal domains. Cell. 1991;64:941-950.

52. Kellum R., Schedl P. A group of scs elements function as domain boundaries in an enhancer-blocking assay. Mol. Cell. Biol. 1992;12: 2424-2431.

53. Kheradpour P., Ernst J., Melnikov A., Rogov P., Wang L., Zhang X., Alston J., Mikkelsen T.S., Kellis M. Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay. Genome Res. 2013;23:800-811.

54. Kim M.J., Ahituv N. The hydrodynamic tail vein assay as a tool for the study of liver promoters and enhancers. Methods Mol. Biol. 2013; 1015:279-289.

55. Kundaje A., Meuleman W., Ernst J., Bilenky M., Yen A., Heravi-Moussavi A., Kheradpour P., …, Hirst M., Meissner A., Milosavljevic A., Ren B., Stamatoyannopoulos J.A., Wang T., Kellis M. Integrative analysis of 111 reference human epigenomes. Nature. 2015;518: 317-330.

56. Kvon E.Z., Kazmar T., Stampfel G., Yanez-Cuna J.O., Pagani M., Schernhuber K., Dickson B.J., Stark A. Genome-scale functional characterization of Drosophila developmental enhancers in vivo. Nature. 2014;512:91-95.

57. Kvon E.Z., Stampfel G., Yanez-Cuna J.O., Dickson B.J., Stark A. HOT regions function as patterned developmental enhancers and have a distinct cis-regulatory signature. Genes Dev. 2012;26:908-913.

58. Kwasnieski J.C., Fiore C., Chaudhari H.G., Cohen B.A. High-throughput functional testing of ENCODE segmentation predictions. Genome Res. 2014;24:1595-1602.

59. 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. 2012;109:19498-19503.

60. Lanzuolo C., Roure V., Dekker J., Bantignies F., Orlando V. Polycomb response elements mediate the formation of chromosome higherorder structures in the bithorax complex. Nat. Cell Biol. 2007;9: 1167-1174.

61. Lettice L.A., Williamson I., Devenney P.S., Kilanowski F., Dorin J., Hill R.E. Development of five digits is controlled by a bipartite longrange cis-regulator. Development. 2014;141:1715-1725.

62. Li K., Liu Y., Cao H., Zhang Y., Gu Z., Liu X., Yu A., Kaphle P., Dickerson K.E., Ni M., Xu J. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nat. Commun. 2020; 11:485.

63. Li L., He S., Sun J.M., Davie J.R. Gene regulation by Sp1 and Sp3. Biochem. Cell Biol. 2004;82:460-471.

64. Lieberman-Aiden E., van Berkum N.L., Williams L., Imakaev M., Ragoczy T., Telling A., Amit I., Lajoie B.R., Sabo P.J., Dorschner M.O., Sandstrom R., Bernstein B., Bender M.A., Groudine M., Gnirke A., Stamatoyannopoulos J., Mirny L.A., Lander E.S., Dekker J. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science. 2009;326:289-293.

65. Manolio T.A., Collins F.S., Cox N.J., Goldstein D.B., Hindorff L.A., Hunter D.J., McCarthy M.I., …, Clark A.G., Eichler E.E., Gibson G., Haines J.L., Mackay T.F., McCarroll S.A., Visscher P.M. Finding the missing heritability of complex diseases. Nature. 2009;461: 747-753.

66. Maricque B.B., Chaudhari H.G., Cohen B.A. A massively parallel reporter assay dissects the influence of chromatin structure on cis-regulatory activity. Nat. Biotechnol. 2018. DOI 10.1038/nbt.4285.

67. Maricque B.B., Dougherty J.D., Cohen B.A. A genome-integrated massively parallel reporter assay reveals DNA sequence determinants of cis-regulatory activity in neural cells. Nucleic Acids Res. 2017; 45:e16.

68. Maurano M.T., Humbert R., Rynes E., Thurman R.E., Haugen E., Wang H., Reynolds A.P., …, Ziegler S., Cotsapas C., Sotoodehnia N., Glass I., Sunyaev S.R., Kaul R., Stamatoyannopoulos J.A. Systematic localization of common disease-associated variation in regulatory DNA. Science. 2012;337:1190-1195.

69. Melnikov A., Murugan A., Zhang X., Tesileanu T., Wang L., Rogov P., Feizi S., Gnirke A., Callan C.G. Jr., Kinney J.B., Kellis M., Lander E.S., Mikkelsen T.S. Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Nat. Biotechnol. 2012;30:271-277.

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

71. Muller H.J. Types of visible variations induced by X-rays in Drosophila. J. Genet. 1930;299-334.

72. Mumbach M.R., Rubin A.J., Flynn R.A., Dai C., Khavari P.A., Greenleaf W.J., Chang H.Y. HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nat. Methods. 2016;13:919-922.

73. Murtha M., Tokcaer-Keskin Z., Tang Z., Strino F., Chen X., Wang Y., Xi X., Basilico C., Brown S., Bonneau R., Kluger Y., Dailey L. FIREWACh: high-throughput functional detection of transcriptional regulatory modules in mammalian cells. Nat. Methods. 2014;11: 559-565.

74. Nagy P.L., Cleary M.L., Brown P.O., Lieb J.D. Genomewide demarcation of RNA polymerase II transcription units revealed by physical fractionation of chromatin. Proc. Natl. Acad. Sci. USA. 2003;100: 6364-6369.

75. Nettling M., Treutler H., Cerquides J., Grosse I. Detecting and correcting the binding-affinity bias in ChIP-seq data using inter-species information. BMC Genomics. 2016;17:347.

76. Nora E.P., Goloborodko A., Valton A.L., Gibcus J.H., Uebersohn A., Abdennur N., Dekker J., Mirny L.A., Bruneau B.G. Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell. 2017;169:930- 944.e922.

77. Pang B., Snyder M.P. Systematic identification of silencers in human cells. Nat. Genet. 2020;52:254-263.

78. Paredes S.H., Melgar M.F., Sethupathy P. Promoter-proximal CCCTCfactor binding is associated with an increase in the transcriptional pausing index. Bioinformatics. 2013;29:1485-1487.

79. Pasquali L., Gaulton K.J., Rodriguez-Segui S.A., Mularoni L., MiguelEscalada I., Akerman I., Tena J.J., …, Berney T., Gloyn A.L., Ravassard P., Skarmeta J.L.G., Muller F., McCarthy M.I., Ferrer J. Pancreatic islet enhancer clusters enriched in type 2 diabetes riskassociated variants. Nat. Genet. 2014;46:136-143.

80. Patwardhan R.P., Hiatt J.B., Witten D.M., Kim M.J., Smith R.P., May D., Lee C., Andrie J.M., Lee S.I., Cooper G.M., Ahituv N., Pennacchio L.A., Shendure J. Massively parallel functional dissection of mammalian enhancers in vivo. Nat. Biotechnol. 2012;30:265-270.

81. Pennacchio L.A., Bickmore W., Dean A., Nobrega M.A., Bejerano G. Enhancers: five essential questions. Nat. Rev. Genet. 2013;14:288- 295.

82. Phillips-Cremins J.E., Corces V.G. Chromatin insulators: linking genome organization to cellular function. Mol. Cell. 2013;50:461-474.

83. Phillips-Cremins J.E., Sauria M.E., Sanyal A., Gerasimova T.I., Lajoie B.R., Bell J.S., Ong C.T., Hookway T.A., Guo C., Sun Y., Bland M.J., Wagstaff W., Dalton S., McDevitt T.C., Sen R., Dekker J., Taylor J., Corces V.G. Architectural protein subclasses shape 3D organization of genomes during lineage commitment. Cell. 2013;153:1281-1295.

84. Rada-Iglesias A., Bajpai R., Swigut T., Brugmann S.A., Flynn R.A., Wysocka J. A unique chromatin signature uncovers early developmental enhancers in humans. Nature. 2011;470:279-283.

85. Rao S.S., Huntley M.H., Durand N.C., Stamenova E.K., Bochkov I.D., Robinson J.T., Sanborn A.L., Machol I., Omer A.D., Lander E.S., Aiden E.L. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014;159:1665- 1680.

86. Rao S.S.P., Huang S.C., Glenn St Hilaire B., Engreitz J.M., Perez E.M., Kieffer-Kwon K.R., Sanborn A.L., Johnstone S.E., Bascom G.D., Bochkov I.D., Huang X., Shamim M.S., Shin J., Turner D., Ye Z., Omer A.D., Robinson J.T., Schlick T., Bernstein B.E., Casellas R., Lander E.S., Aiden E.L. Cohesin loss eliminates all loop domains. Cell. 2017;171:305-320 e324.

87. Rye M.B., Saetrom P., Drablos F. A manually curated ChIP-seq benchmark demonstrates room for improvement in current peak-finder programs. Nucleic Acids Res. 2011;39:e25. DOI 10.1093/nar/gkq1187.

88. Sandmann T., Girardot C., Brehme M., Tongprasit W., Stolc V., Furlong E.E. A core transcriptional network for early mesoderm development in Drosophila melanogaster. Genes Dev. 2007;21: 436-449.

89. Sanjana N.E., Wright J., Zheng K., Shalem O., Fontanillas P., Joung J., Cheng C., Regev A., Zhang F. High-resolution interrogation of functional elements in the noncoding genome. Science. 2016;353:1545- 1549.

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

91. Shukla S., Kavak E., Gregory M., Imashimizu M., Shutinoski B., Kashlev M., Oberdoerffer P., Sandberg R., Oberdoerffer S. CTCFpromoted RNA polymerase II pausing links DNA methylation to splicing. Nature. 2011;479:74-79.

92. Skene P.J., Henikoff S. An efficient targeted nuclease strategy for highresolution mapping of DNA binding sites. eLife. 2017;6:e21856. DOI 10.7554/eLife.21856.

93. Song L., Crawford G.E. DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells. Cold Spring Harb. Protoc. 2010;2010(2):pdbprot 5384.

94. Song L., Zhang Z., Grasfeder L.L., Boyle A.P., Giresi P.G., Lee B.K., Sheffield N.C., …, Winter D., Clarke N.D., Birney E., Iyer V.R.,Crawford G.E., Lieb J.D., Furey T.S. Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape celltype identity. Genome Res. 2011;21:1757-1767.

95. Srinivasan L., Atchison M.L. YY1 DNA binding and PcG recruitment requires CtBP. Genes Dev. 2004;18:2596-2601.

96. Stitzel M.L., Sethupathy P., Pearson D.S., Chines P.S., Song L., Erdos M.R., Welch R., Parker S.C., Boyle A.P., Scott L.J., Margulies E.H., Boehnke M., Furey T.S., Crawford G.E., Collins F.S. Global epigenomic analysis of primary human pancreatic islets provides insights into type 2 diabetes susceptibility loci. Cell Metab. 2010;12:443-455.

97. Sur I., Taipale J. The role of enhancers in cancer. Nat. Rev. Cancer. 2016;16:483-493.

98. Tiwari V.K., McGarvey K.M., Licchesi J.D., Ohm J.E., Herman J.G., Schubeler D., Baylin S.B. PcG proteins, DNA methylation, and gene repression by chromatin looping. PLoS Biol. 2008;6:2911-2927.

99. Tolhuis B., Palstra R.J., Splinter E., Grosveld F., de Laat W. Looping and interaction between hypersensitive sites in the active beta-globin locus. Mol. Cell. 2002;10:1453-1465.

100. Trynka G., Sandor C., Han B., Xu H., Stranger B.E., Liu X.S., Raychaudhuri S. Chromatin marks identify critical cell types for fine mapping complex trait variants. Nat. Genet. 2013;45:124-130.

101. Visel A., Blow M.J., Li Z., Zhang T., Akiyama J.A., Holt A., PlajzerFrick I., Shoukry M., Wright C., Chen F., Afzal V., Ren B., Rubin E.M., Pennacchio L.A. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature. 2009;457:854-858.

102. Whyte W.A., Orlando D.A., Hnisz D., Abraham B.J., Lin C.Y., Kagey M.H., Rahl P.B., Lee T.I., Young R.A. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell. 2013;153:307-319.

103. Yang J., Corces V.G. Chromatin insulators: a role in nuclear organization and gene expression. Adv. Cancer Res. 2011;110:43-76.

104. Yang S.H., Cheng P.H., Sullivan R.T., Thomas J.W., Chan A.W. Lentiviral integration preferences in transgenic mice. Genesis. 2008;46: 711-718.

105. Zabidi M.A., Arnold C.D., Schernhuber K., Pagani M., Rath M., Frank O., Stark A. Enhancer-core-promoter specificity separates developmental and housekeeping gene regulation. Nature. 2015;518: 556-559.