Subtle Changes in Motif Positioning Cause Tissue-Specific Effects on Robustness of an Enhancer's Activity

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Deciphering the specific contribution of individual motifs within cis-regulatory modules (CRMs) is crucial to understanding how gene expression is regulated and how this process is affected by sequence variation. But despite vast improvements in the ability to identify where transcription factors (TFs) bind throughout the genome, we are limited in our ability to relate information on motif occupancy to function from sequence alone. Here, we engineered 63 synthetic CRMs to systematically assess the relationship between variation in the content and spacing of motifs within CRMs to CRM activity during development using Drosophila transgenic embryos. In over half the cases, very simple elements containing only one or two types of TF binding motifs were capable of driving specific spatio-temporal patterns during development. Different motif organizations provide different degrees of robustness to enhancer activity, ranging from binary on-off responses to more subtle effects including embryo-to-embryo and within-embryo variation. By quantifying the effects of subtle changes in motif organization, we were able to model biophysical rules that explain CRM behavior and may contribute to the spatial positioning of CRM activity in vivo. For the same enhancer, the effects of small differences in motif positions varied in developmentally related tissues, suggesting that gene expression may be more susceptible to sequence variation in one tissue compared to another. This result has important implications for human eQTL studies in which many associated mutations are found in cis-regulatory regions, though the mechanism for how they affect tissue-specific gene expression is often not understood.

Vyšlo v časopise: Subtle Changes in Motif Positioning Cause Tissue-Specific Effects on Robustness of an Enhancer's Activity. PLoS Genet 10(1): e32767. doi:10.1371/journal.pgen.1004060
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004060

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1. SpitzF, FurlongEE (2012) Transcription factors: from enhancer binding to developmental control. Nat Rev Genet 13: 613–626.

2. JunionG, SpivakovM, GirardotC, BraunM, GustafsonEH, et al. (2012) A transcription factor collective defines cardiac cell fate and reflects lineage history. Cell 148: 473–486.

3. LiXY, MacArthurS, BourgonR, NixD, PollardDA, et al. (2008) Transcription factors bind thousands of active and inactive regions in the Drosophila blastoderm. PLoS Biol 6: e27.

4. MacArthurS, LiXY, LiJ, BrownJB, ChuHC, et al. (2009) Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions. Genome Biol 10: R80.

5. SandmannT, GirardotC, BrehmeM, TongprasitW, StolcV, et al. (2007) A core transcriptional network for early mesoderm development in Drosophila melanogaster. Genes Dev 21: 436–449.

6. ZeitlingerJ, ZinzenRP, StarkA, KellisM, ZhangH, et al. (2007) Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo. Genes Dev 21: 385–390.

7. ZinzenRP, GirardotC, GagneurJ, BraunM, FurlongEE (2009) Combinatorial binding predicts spatio-temporal cis-regulatory activity. Nature 462: 65–70.

8. ViselA, BlowMJ, LiZ, ZhangT, AkiyamaJA, et al. (2009) ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457: 854–858.

9. BlowMJ, McCulleyDJ, LiZ, ZhangT, AkiyamaJA, et al. (2010) ChIP-Seq identification of weakly conserved heart enhancers. Nat Genet 42: 806–810.

10. BernsteinBE, BirneyE, DunhamI, GreenED, GunterC, et al. (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489: 57–74.

11. BonnS, ZinzenRP, GirardotC, GustafsonEH, Perez-GonzalezA, et al. (2012) Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic development. Nat Genet 44: 148–156.

12. ErnstJ, KheradpourP, MikkelsenTS, ShoreshN, WardLD, et al. (2011) Mapping and analysis of chromatin state dynamics in nine human cell types. Nature 473: 43–49.

13. HeintzmanND, HonGC, HawkinsRD, KheradpourP, StarkA, et al. (2009) Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459: 108–112.

14. HeintzmanND, StuartRK, HonG, FuY, ChingCW, et al. (2007) Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet 39: 311–318.

15. RoyS, ErnstJ, KharchenkoPV, KheradpourP, NegreN, et al. (2010) Identification of functional elements and regulatory circuits by Drosophila modENCODE. Science 330: 1787–1797.

16. BusserBW, HuangD, RogackiKR, LaneEA, ShokriL, et al. (2012) Integrative analysis of the zinc finger transcription factor Lame duck in the Drosophila myogenic gene regulatory network. Proc Natl Acad Sci U S A 109: 20768–20773.

17. HalfonMS, CarmenaA, GisselbrechtS, SackersonCM, JimenezF, et al. (2000) Ras pathway specificity is determined by the integration of multiple signal-activated and tissue-restricted transcription factors. Cell 103: 63–74.

18. IpYT, ParkRE, KosmanD, YazdanbakhshK, LevineM (1992) dorsal-twist interactions establish snail expression in the presumptive mesoderm of the Drosophila embryo. Genes Dev 6: 1518–1530.

19. StruffiP, CoradoM, KaplanL, YuD, RushlowC, et al. (2011) Combinatorial activation and concentration-dependent repression of the Drosophila even skipped stripe 3+7 enhancer. Development 138: 4291–4299.

20. SwansonCI, EvansNC, BaroloS (2010) Structural rules and complex regulatory circuitry constrain expression of a Notch- and EGFR-regulated eye enhancer. Dev Cell 18: 359–370.

21. D'AlonzoRC, SelvamuruganN, KarsentyG, PartridgeNC (2002) Physical interaction of the activator protein-1 factors c-Fos and c-Jun with Cbfa1 for collagenase-3 promoter activation. J Biol Chem 277: 816–822.

22. ErivesA, LevineM (2004) Coordinate enhancers share common organizational features in the Drosophila genome. Proc Natl Acad Sci U S A 101: 3851–3856.

23. InoueJ, SatoR, MaedaM (1998) Multiple DNA elements for sterol regulatory element-binding protein and NF-Y are responsible for sterol-regulated transcription of the genes for human 3-hydroxy-3-methylglutaryl coenzyme A synthase and squalene synthase. J Biochem 123: 1191–1198.

24. PanneD, ManiatisT, HarrisonSC (2007) An atomic model of the interferon-beta enhanceosome. Cell 129: 1111–1123.

25. ThanosD, ManiatisT (1995) Virus induction of human IFN beta gene expression requires the assembly of an enhanceosome. Cell 83: 1091–1100.

26. ZinzenRP, SengerK, LevineM, PapatsenkoD (2006) Computational models for neurogenic gene expression in the Drosophila embryo. Curr Biol 16: 1358–1365.

27. JinH, StojnicR, AdryanB, OzdemirA, StathopoulosA, et al. (2013) Genome-wide screens for in vivo Tinman binding sites identify cardiac enhancers with diverse functional architectures. PLoS Genet 9: e1003195.

28. BrownCD, JohnsonDS, SidowA (2007) Functional architecture and evolution of transcriptional elements that drive gene coexpression. Science 317: 1557–1560.

29. LibermanLM, StathopoulosA (2009) Design flexibility in cis-regulatory control of gene expression: synthetic and comparative evidence. Dev Biol 327: 578–589.

30. RastegarS, HessI, DickmeisT, NicodJC, ErtzerR, et al. (2008) The words of the regulatory code are arranged in a variable manner in highly conserved enhancers. Dev Biol 318: 366–377.

31. SwansonCI, SchwimmerDB, BaroloS (2011) Rapid evolutionary rewiring of a structurally constrained eye enhancer. Curr Biol 21: 1186–1196.

32. ParkerDS, WhiteMA, RamosAI, CohenBA, BaroloS (2011) The cis-regulatory logic of Hedgehog gradient responses: key roles for gli binding affinity, competition, and cooperativity. Sci Signal 4: ra38.

33. BullaugheyK (2011) Changes in selective effects over time facilitate turnover of enhancer sequences. Genetics 187: 567–582.

34. GertzJ, SiggiaED, CohenBA (2009) Analysis of combinatorial cis-regulation in synthetic and genomic promoters. Nature 457: 215–218.

35. SharonE, KalmaY, SharpA, Raveh-SadkaT, LevoM, et al. (2012) Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters. Nat Biotechnol 30: 521–530.

36. MelnikovA, MuruganA, ZhangX, TesileanuT, WangL, et al. (2012) Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Nat Biotechnol 30: 271–277.

37. PatwardhanRP, HiattJB, WittenDM, KimMJ, SmithRP, et al. (2012) Massively parallel functional dissection of mammalian enhancers in vivo. Nat Biotechnol 30: 265–270.

38. ZichnerT, GarfieldDA, RauschT, StutzAM, CannavoE, et al. (2013) Impact of genomic structural variation in Drosophila melanogaster based on population-scale sequencing. Genome research 23: 568–579.

39. MassourasA, WaszakSM, Albarca-AguileraM, HensK, HolcombeW, et al. (2012) Genomic variation and its impact on gene expression in Drosophila melanogaster. PLoS genetics 8: e1003055.

40. WeischenfeldtJ, SymmonsO, SpitzF, KorbelJO (2013) Phenotypic impact of genomic structural variation: insights from and for human disease. Nat Rev Genet 14: 125–138.

41. BonnS, FurlongEE (2008) cis-Regulatory networks during development: a view of Drosophila. Curr Opin Genet Dev 18: 513–520.

42. CiglarL, FurlongEE (2009) Conservation and divergence in developmental networks: a view from Drosophila myogenesis. Curr Opin Cell Biol 21: 754–760.

43. BischofJ, MaedaRK, HedigerM, KarchF, BaslerK (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci U S A 104: 3312–3317.

44. DavidsonEH (2010) Emerging properties of animal gene regulatory networks. Nature 468: 911–920.

45. LifanovAP, MakeevVJ, NazinaAG, PapatsenkoDA (2003) Homotypic regulatory clusters in Drosophila. Genome Res 13: 579–588.

46. GoteaV, ViselA, WestlundJM, NobregaMA, PennacchioLA, et al. (2010) Homotypic clusters of transcription factor binding sites are a key component of human promoters and enhancers. Genome Res 20: 565–577.

47. HanesSD, RiddihoughG, Ish-HorowiczD, BrentR (1994) Specific DNA recognition and intersite spacing are critical for action of the bicoid morphogen. Mol Cell Biol 14: 3364–3375.

48. Simpson-BroseM, TreismanJ, DesplanC (1994) Synergy between the hunchback and bicoid morphogens is required for anterior patterning in Drosophila. Cell 78: 855–865.

49. SzymanskiP, LevineM (1995) Multiple modes of dorsal-bHLH transcriptional synergy in the Drosophila embryo. EMBO J 14: 2229–2238.

50. FulkersonE, EstesPA (2010) Common motifs shared by conserved enhancers of Drosophila midline glial genes. J Exp Zool B Mol Dev Evol 316: 61–75.

51. RothbacherU, BertrandV, LamyC, LemaireP (2007) A combinatorial code of maternal GATA, Ets and beta-catenin-TCF transcription factors specifies and patterns the early ascidian ectoderm. Development 134: 4023–4032.

52. VincentJP, KassisJA, O'FarrellPH (1990) A synthetic homeodomain binding site acts as a cell type specific, promoter specific enhancer in Drosophila embryos. EMBO J 9: 2573–2578.

53. BaroloS (2006) Transgenic Wnt/TCF pathway reporters: all you need is Lef? Oncogene 25: 7505–7511.

54. RieseJ, YuX, MunnerlynA, EreshS, HsuSC, et al. (1997) LEF-1, a nuclear factor coordinating signaling inputs from wingless and decapentaplegic. Cell 88: 777–787.

55. BaroloS, WalkerRG, PolyanovskyAD, FreschiG, KeilT, et al. (2000) A notch-independent activity of suppressor of hairless is required for normal mechanoreceptor physiology. Cell 103: 957–969.

56. GussKA, NelsonCE, HudsonA, KrausME, CarrollSB (2001) Control of a genetic regulatory network by a selector gene. Science 292: 1164–1167.

57. ZaffranS, KuchlerA, LeeHH, FraschM (2001) biniou (FoxF), a central component in a regulatory network controlling visceral mesoderm development and midgut morphogenesis in Drosophila. Genes Dev 15: 2900–2915.

58. JakobsenJS, BraunM, AstorgaJ, GustafsonEH, SandmannT, et al. (2007) Temporal ChIP-on-chip reveals Biniou as a universal regulator of the visceral muscle transcriptional network. Genes Dev 21: 2448–2460.

59. SunB, HurshDA, JacksonD, BeachyPA (1995) Ultrabithorax protein is necessary but not sufficient for full activation of decapentaplegic expression in the visceral mesoderm. EMBO J 14: 520–535.

60. YangX, van BeestM, CleversH, JonesT, HurshDA, et al. (2000) decapentaplegic is a direct target of dTcf repression in the Drosophila visceral mesoderm. Development 127: 3695–3702.

61. MartyT, MullerB, BaslerK, AffolterM (2000) Schnurri mediates Dpp-dependent repression of brinker transcription. Nat Cell Biol 2: 745–749.

62. SallerE, BienzM (2001) Direct competition between Brinker and Drosophila Mad in Dpp target gene transcription. EMBO Rep 2: 298–305.

63. MullenAC, OrlandoDA, NewmanJJ, LovenJ, KumarRM, et al. (2011) Master transcription factors determine cell-type-specific responses to TGF-beta signaling. Cell 147: 565–576.

64. TrompoukiE, BowmanTV, LawtonLN, FanZP, WuDC, et al. (2011) Lineage regulators direct BMP and Wnt pathways to cell-specific programs during differentiation and regeneration. Cell 147: 577–589.

65. NewfeldSJ, ChartoffEH, GraffJM, MeltonDA, GelbartWM (1996) Mothers against dpp encodes a conserved cytoplasmic protein required in DPP/TGF-beta responsive cells. Development 122: 2099–2108.

66. ImmergluckK, LawrencePA, BienzM (1990) Induction across germ layers in Drosophila mediated by a genetic cascade. Cell 62: 261–268.

67. FernandezBG, AriasAM, JacintoA (2007) Dpp signalling orchestrates dorsal closure by regulating cell shape changes both in the amnioserosa and in the epidermis. Mech Dev 124: 884–897.

68. BruneauBG, NemerG, SchmittJP, CharronF, RobitailleL, et al. (2001) A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106: 709–721.

69. DurocherD, CharronF, WarrenR, SchwartzRJ, NemerM (1997) The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors. EMBO J 16: 5687–5696.

70. GajewskiK, ZhangQ, ChoiCY, FossettN, DangA, et al. (2001) Pannier is a transcriptional target and partner of Tinman during Drosophila cardiogenesis. Dev Biol 233: 425–436.

71. ZaffranS, XuX, LoPC, LeeHH, FraschM (2002) Cardiogenesis in the Drosophila model: control mechanisms during early induction and diversification of cardiac progenitors. Cold Spring Harb Symp Quant Biol 67: 1–12.

72. CrippsRM, OlsonEN (2002) Control of cardiac development by an evolutionarily conserved transcriptional network. Dev Biol 246: 14–28.

73. BodmerR (1993) The gene tinman is required for specification of the heart and visceral muscles in Drosophila. Development 118: 719–729.

74. AzpiazuN, FraschM (1993) tinman and bagpipe: two homeo box genes that determine cell fates in the dorsal mesoderm of Drosophila. Genes Dev 7: 1325–1340.

75. ReimI, FraschM (2005) The Dorsocross T-box genes are key components of the regulatory network controlling early cardiogenesis in Drosophila. Development 132: 4911–4925.

76. GajewskiK, FossettN, MolkentinJD, SchulzRA (1999) The zinc finger proteins Pannier and GATA4 function as cardiogenic factors in Drosophila. Development 126: 5679–5688.

77. FraschM (1995) Induction of visceral and cardiac mesoderm by ectodermal Dpp in the early Drosophila embryo. Nature 374: 464–467.

78. LockwoodWK, BodmerR (2002) The patterns of wingless, decapentaplegic, and tinman position the Drosophila heart. Mech Dev 114: 13–26.

79. HerranzH, MorataG (2001) The functions of pannier during Drosophila embryogenesis. Development 128: 4837–4846.

80. KlinedinstSL, BodmerR (2003) Gata factor Pannier is required to establish competence for heart progenitor formation. Development 130: 3027–3038.

81. GibsonG (1996) Epistasis and pleiotropy as natural properties of transcriptional regulation. Theor Popul Biol 49: 58–89.

82. AckersGK, JohnsonAD, SheaMA (1982) Quantitative model for gene regulation by lambda phage repressor. Proc Natl Acad Sci U S A 79: 1129–1133.

83. AmitR, GarciaHG, PhillipsR, FraserSE (2011) Building enhancers from the ground up: a synthetic biology approach. Cell 146: 105–118.

84. LaikenSL, GrossCA, Von HippelPH (1972) Equilibrium and kinetic studies of Escherichia coli lac repressor-inducer interactions. J Mol Biol 66: 143–155.

85. OppenheimAB, KobilerO, StavansJ, CourtDL, AdhyaS (2005) Switches in bacteriophage lambda development. Annu Rev Genet 39: 409–429.

86. PtashneM (2005) Regulation of transcription: from lambda to eukaryotes. Trends Biochem Sci 30: 275–279.

87. FakhouriWD, AyA, SayalR, DreschJ, DayringerE, et al. (2010) Deciphering a transcriptional regulatory code: modeling short-range repression in the Drosophila embryo. Mol Syst Biol 6: 341.

88. CrockerJ, TamoriY, ErivesA (2008) Evolution acts on enhancer organization to fine-tune gradient threshold readouts. PLoS Biol 6: e263.

89. KnirrS, FraschM (2001) Molecular integration of inductive and mesoderm-intrinsic inputs governs even-skipped enhancer activity in a subset of pericardial and dorsal muscle progenitors. Dev Biol 238: 13–26.

90. BabuRajendranN, PalasingamP, NarasimhanK, SunW, PrabhakarS, et al. (2010) Structure of Smad1 MH1/DNA complex reveals distinctive rearrangements of BMP and TGF-beta effectors. Nucleic Acids Res 38: 3477–3488.

91. LaRonde-LeBlancNA, WolbergerC (2003) Structure of HoxA9 and Pbx1 bound to DNA: Hox hexapeptide and DNA recognition anterior to posterior. Genes Dev 17: 2060–2072.

92. XuX, YinZ, HudsonJB, FergusonEL, FraschM (1998) Smad proteins act in combination with synergistic and antagonistic regulators to target Dpp responses to the Drosophila mesoderm. Genes Dev 12: 2354–2370.

93. BoettigerAN, LevineM (2009) Synchronous and stochastic patterns of gene activation in the Drosophila embryo. Science 325: 471–473.

94. LevineM (2011) Paused RNA polymerase II as a developmental checkpoint. Cell 145: 502–511.

95. PerryMW, BoettigerAN, BothmaJP, LevineM (2010) Shadow enhancers foster robustness of Drosophila gastrulation. Curr Biol 20: 1562–1567.

96. HongJW, HendrixDA, LevineMS (2008) Shadow enhancers as a source of evolutionary novelty. Science 321: 1314.

97. PerryMW, CandeJD, BoettigerAN, LevineM (2009) Evolution of insect dorsoventral patterning mechanisms. Cold Spring Harb Symp Quant Biol 74: 275–279.

98. FrankelN, DavisGK, VargasD, WangS, PayreF, et al. (2010) Phenotypic robustness conferred by apparently redundant transcriptional enhancers. Nature 466: 490–493.

99. LudwigMZ, Manu, KittlerR, WhiteKP, KreitmanM (2011) Consequences of eukaryotic enhancer architecture for gene expression dynamics, development, and fitness. PLoS Genet 7: e1002364.

100. BaroloS, CarverLA, PosakonyJW (2000) GFP and beta-galactosidase transformation vectors for promoter/enhancer analysis in Drosophila. Biotechniques 29: 726, 728, 730, 732.

101. FurlongEE, AndersenEC, NullB, WhiteKP, ScottMP (2001) Patterns of gene expression during Drosophila mesoderm development. Science 293: 1629–1633.

102. AyA, ArnostiDN (2011) Mathematical modeling of gene expression: a guide for the perplexed biologist. Crit Rev Biochem Mol Biol 46: 137–151.