Consequences of Eukaryotic Enhancer Architecture for Gene Expression Dynamics, Development, and Fitness
The regulatory logic of time- and tissue-specific gene expression has mostly been dissected in the context of the smallest DNA fragments that, when isolated, recapitulate native expression in reporter assays. It is not known if the genomic sequences surrounding such fragments, often evolutionarily conserved, have any biological function or not. Using an enhancer of the even-skipped gene of Drosophila as a model, we investigate the functional significance of the genomic sequences surrounding empirically identified enhancers. A 480 bp long “minimal stripe element” is able to drive even-skipped expression in the second of seven stripes but is embedded in a larger region of 800 bp containing evolutionarily conserved binding sites for required transcription factors. To assess the overall fitness contribution made by these binding sites in the native genomic context, we employed a gene-replacement strategy in which whole-locus transgenes, capable of rescuing even-skipped- lethality to adulthood, were substituted for the native gene. The molecular phenotypes were characterized by tagging Even-skipped with a fluorescent protein and monitoring gene expression dynamics in living embryos. We used recombineering to excise the sequences surrounding the minimal enhancer and site-specific transgenesis to create co-isogenic strains differing only in their stripe 2 sequences. Remarkably, the flanking sequences were dispensable for viability, proving the sufficiency of the minimal element for biological function under normal conditions. These sequences are required for robustness to genetic and environmental perturbation instead. The mutant enhancers had measurable sex- and dose-dependent effects on viability. At the molecular level, the mutants showed a destabilization of stripe placement and improper activation of downstream genes. Finally, we demonstrate through live measurements that the peripheral sequences are required for temperature compensation. These results imply that seemingly redundant regulatory sequences beyond the minimal enhancer are necessary for robust gene expression and that “robustness” itself must be an evolved characteristic of the wild-type enhancer.
Vyšlo v časopise:
Consequences of Eukaryotic Enhancer Architecture for Gene Expression Dynamics, Development, and Fitness. PLoS Genet 7(11): e32767. doi:10.1371/journal.pgen.1002364
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1002364
Souhrn
The regulatory logic of time- and tissue-specific gene expression has mostly been dissected in the context of the smallest DNA fragments that, when isolated, recapitulate native expression in reporter assays. It is not known if the genomic sequences surrounding such fragments, often evolutionarily conserved, have any biological function or not. Using an enhancer of the even-skipped gene of Drosophila as a model, we investigate the functional significance of the genomic sequences surrounding empirically identified enhancers. A 480 bp long “minimal stripe element” is able to drive even-skipped expression in the second of seven stripes but is embedded in a larger region of 800 bp containing evolutionarily conserved binding sites for required transcription factors. To assess the overall fitness contribution made by these binding sites in the native genomic context, we employed a gene-replacement strategy in which whole-locus transgenes, capable of rescuing even-skipped- lethality to adulthood, were substituted for the native gene. The molecular phenotypes were characterized by tagging Even-skipped with a fluorescent protein and monitoring gene expression dynamics in living embryos. We used recombineering to excise the sequences surrounding the minimal enhancer and site-specific transgenesis to create co-isogenic strains differing only in their stripe 2 sequences. Remarkably, the flanking sequences were dispensable for viability, proving the sufficiency of the minimal element for biological function under normal conditions. These sequences are required for robustness to genetic and environmental perturbation instead. The mutant enhancers had measurable sex- and dose-dependent effects on viability. At the molecular level, the mutants showed a destabilization of stripe placement and improper activation of downstream genes. Finally, we demonstrate through live measurements that the peripheral sequences are required for temperature compensation. These results imply that seemingly redundant regulatory sequences beyond the minimal enhancer are necessary for robust gene expression and that “robustness” itself must be an evolved characteristic of the wild-type enhancer.
Zdroje
1. SchaffnerW 1999 Enhancer. CreightonT The Encyclopedia of Molecular Biology New York John Wiley and Sons, Inc. 823 828
2. PennacchioLARubinEM 2001 Genomic strategies to identify mammalian regulatory sequences. Nat Rev Genet 2 100 109
3. LudwigMZ 2002 Functional evolution of noncoding DNA. Curr Opin Genet Dev 12 634 639
4. PierstorffNBergmanCMWieheT 2006 Identifying cis-regulatory modules by combining comparative and compositional analysis of DNA. Bioinformatics 22 2858 2864
5. HeXLingXSinhaS 2009 Alignment and prediction of cis-regulatory modules based on a probabilistic model of evolution. PLoS Comput Biol 5 e1000299 doi:10.1371/journal.pcbi.1000299
6. SuJTeichmannSADownTA 2010 Assessing computational methods of cis-regulatory module prediction. PLoS Comput Biol 6 e1001020 doi:10.1371/journal.pcbi.1001020
7. ArnoneMIDavidsonEH 1997 The hardwiring of development: organization and function of genomic regulatory systems. Development 124 1851 1864
8. SodergrenEWeinstockGMDavidsonEHCameronRAGibbsRA 2006 The genome of the sea urchin Strongylocentrotus purpuratus. Science 314 941 952
9. BermanBPNibuYPfeifferBDTomancakPCelnikerSE 2002 Exploiting transcription factor binding site clustering to identify cis-regulatory modules involved in pattern formation in the Drosophila genome. Proc Natl Acad Sci U S A 99 757 762
10. BermanBPPfeifferBDLavertyTRSalzbergSLRubinGM 2004 Computational identification of developmental enhancers: conservation and function of transcription factor binding-site clusters in Drosophila melanogaster and Drosophila pseudoobscura. Genome Biology 5 R61
11. ErivesALevineM 2004 Coordinate enhancers share common organizational features in the Drosophila genome. Proc Natl Acad Sci U S A 101 3851 3856
12. HalfonMSGradYChurchGMMichelsonAM 2002 Computation-based discovery of related transcriptional regulatory modules and motifs using an experimentally validated combinatorial model. Genome Res 12 1019 1028
13. LiLZhuQHeXSinhaSHalfonMS 2007 Large-scale analysis of transcriptional cis-regulatory modules reveals both common features and distinct subclasses. Genome Biol 8 R101
14. BergOGvon HippelPH 1987 Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters. J Mol Biol 193 723 750
15. StormoGD 2000 DNA binding sites: representation and discovery. Bioinformatics 16 16 23
16. TanayA 2006 Extensive low-affinity transcriptional interactions in the yeast genome. Genome Res 16 962 972
17. JanssensHHouSJaegerJKimARMyasnikovaE 2006 Quantitative and predictive model of transcriptional control of the Drosophila melanogaster even skipped gene. Nat Genet 38 1159 1165
18. SegalERaveh-SadkaTSchroederMUnnerstallUGaulU 2008 Predicting expression patterns from regulatory sequence in Drosophila segmentation. Nature 451 535 540
19. KazemianMBlattiCRichardsAMcCutchanMWakabayashi-ItoN 2010 Quantitative analysis of the Drosophila segmentation regulatory network using pattern generating potentials. PLoS Biol 8 e1000456 doi:10.1371/journal.pbio.1000456
20. HardingKHoeyTWarriorRLevineM 1989 Autoregulatory and gap gene response elements of the even-skipped promoter of Drosophila. The EMBO Journal 8 1205 1212
21. SmallSBlairALevineM 1996 Regulation of two pair-rule stripes by a single enhancer in the Drosophila embryo. Developmental Biology 175 314 324
22. McGregorAPOrgogozoVDelonIZanetJSrinivasanDG 2007 Morphological evolution through multiple cis-regulatory mutations at a single gene. Nature 448 587 590
23. SchroederMDPearceMFakJFanHUnnerstallU 2004 Transcriptional control in the segmentation gene network of Drosophila. PLoS Biol 2 e271 doi:10.1371/journal.pbio.0020271
24. MosesAMPollardDANixDAIyerVNLiXY 2006 Large-scale turnover of functional transcription factor binding sites in Drosophila. PLoS Comput Biol 2 e130 doi:10.1371/journal.pcbi.0020130
25. HeBZHollowayAKMerklSJKreitmanM 2011 Does positive selection drive transcription factor binding site turnover? A test with Drosophila cis-regulatory modules. PLoS Genet 7 e1002053 doi:10.1371/journal.pgen.1002053
26. LudwigMZPalssonAAlekseevaEBergmanCMNathanJ 2005 Functional evolution of a cis-regulatory module. PLoS Biol 3 e93 doi:10.1371/journal.pbio.0030093
27. LudwigMZPatelNHKreitmanM 1998 Functional analysis of eve stripe 2 enhancer evolution in Drosophila: rules governing conservation and change. Development 125 949 958
28. GotoTMacDonaldPManiatisT 1989 Early and late periodic patterns of even-skipped expression are controlled by distinct regulatory elements that respond to different spatial cues. Cell 57 413 422
29. StanojevicDHoeyTLevineM 1989 Sequence-specific DNA-binding activities of the gap proteins encoded by hunchback and Krüppel in Drosophila. Nature 341 331 335
30. SmallSKrautRHoeyTWarriorRLevineM 1991 Transcriptional regulation of a pair-rule stripe in Drosophila. Genes and Development 5 827 839
31. StanojevicDSmallSLevineM 1991 Regulation of a segmentation stripe by overlapping activators and repressors in the Drosophila embryo. Science 254 1385 1387
32. SmallSBlairALevineM 1992 Regulation of even-skipped stripe 2 in the Drosophila embryo. The EMBO Journal 11 4047 4057
33. ArnostiDNBaroloSLevineMSmallS 1996 The eve stripe 2 enhancer employs multiple modes of transcriptional synergy. Development 122 205 214
34. HouchmandzadehBWieschausELeiblerS 2002 Establishment of developmental precision and proportions in the early Drosophila embryo. Nature 415 798 802
35. GregorTTankDWWieschausEFBialekW 2007 Probing the limits to positional information. Cell 130 153 164
36. SurkovaSKosmanDKozlovKManuMyasnikovaE 2008 Characterization of the Drosophila Segment Determination Morphome. Developmental Biology 313 844 862
37. ElowitzMBLevineAJSiggiaEDSwainPS 2002 Stochastic gene expression in a single cell. Science 297 1183 1186
38. BlakeWJKaernMCantorCRCollinsJJ 2003 Noise in eukaryotic gene expression. Nature 422 633 637
39. AriasAMHaywardP 2006 Filtering transcriptional noise during development: concepts and mechanisms. Nature Reviews Genetics 7 34 44
40. EldarADorfmanRWeissDAsheHShiloBZ 2002 Robustness of the BMP morphogen gradient in Drosophila embryonic patterning. Nature 419 304 308
41. EldarAElowitzMB 2010 Functional roles for noise in genetic circuits. Nature 467 167 173
42. LottSEKreitmanMPalssonAAlekseevaELudwigMZ 2007 Canalization of segmentation and its evolution in Drosophila. Proc Natl Acad Sci U S A 104 10926 10931
43. MilesCMLottSEHendriksCLLudwigMZManu 2010 Artificial selection on egg size perturbs early pattern formation in Drosophila melanogaster. Evolution 65 33 42
44. CheungDMilesCKreitmanMMaJ 2011 Scaling of the Bicoid morphogen gradient by a volume-dependent production rate. Development 138 13 2741 9
45. LucchettaEMLeeJHFuLAPatelNHIsmagilovRF 2005 Dynamics of Drosophila Embryonic Patterning Network Perturbed in Space and Time Using Microfluidics. Nature 434 1134 1138
46. ForceAGCreskoWAPickettFB 2004 Information Accretion, Gene Duplication, and the Mechanisms of Genetic Module Parcellation. SchlosserGWagnerGP Modularity in Development and Evolution Chicago University of Chicago Press 315 337
47. FujiokaMEmi-SarkerYYusibovaGLGotoTJaynesJB 1999 Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients. Development 126 2527 2538
48. GrothACFishMNusseRCalosMP 2004 Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166 1775 1782
49. MarksteinMPitsouliCVillaltaCCelnikerSEPerrimonN 2008 Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nat Genet 40 476 483
50. FujiokaMYusibovaGLPatelNHBrownSJJaynesJB 2002 The repressor activity of Even-skipped is highly conserved, and is sufficient to activate engrailed and to regulate both the spacing and stability of parasegment boundaries. Development 129 4411 4421
51. Nüsslein-VolhardCKludingHJurgensG 1985 Genes affecting the segmental subdivision of the Drosophila embryo. Cold Spring Harbor Symposiums on Quantitative Biology 50 145 154
52. GregorTWieschausEFMcGregorAPBialekWTankDW 2007 Stability and nuclear dynamics of the Bicoid morphogen gradient. Cell 130 141 152
53. InghamPWBakerNEMartinez-AriasA 1988 Regulation of segment polarity genes in the Drosophila blastoderm by fushi-tarazu and even-skipped. Nature 331 73 75
54. FraschMHoeyTRushlowCDoyleHJLevineM 1987 Characterization and localization of the Even-skipped protein of Drosophila. The EMBO Journal 6 749 759
55. HeimRPrasherDCTsienRY 1994 Wavelength mutations and posttranslational autoxidation of Green Fluorescent Protein. Proc Natl Acad Sci U S A 91 12501 12504
56. ManuSurkovaSSpirovAVGurskyVVJanssensH 2009 Canalization of gene expression and domain shifts in the Drosophila blastoderm by dynamical attractors. PLoS Comput Biol 5 e1000303 doi:10.1371/journal.pcbi.1000303
57. SegelIH 1975 Enzyme Kinetics. New York John Wiley & Sons
58. FederMECartanoNVMilosLKrebsRALindquistSL 1996 Effect of engineering Hsp70 copy number on Hsp70 expression and tolerance of ecologically relevant heat shock in larvae and pupae of Drosophila melanogaster. J Exp Biol 199 1837 1844
59. WaddingtonCH 1942 Canalization of development and the inheritance of acquired characters. Nature 150 563 565
60. WaddingtonCH 1959 Canalization of development and genetic assimilation of acquired characters. Nature 183 1654 1655
61. ManuSurkovaSSpirovAVGurskyVVJanssensH 2009 Canalization of gene expression in the Drosophila blastoderm by gap gene cross regulation. PLoS Biol 7 e1000049 doi:10.1371/journal.pbio.1000049
62. HareEEPetersonBKIyerVNMeierREisenMB 2008 Sepsid even-skipped Enhancers Are Functionally Conserved in Drosophila Despite Lack of Sequence Conservation. PLoS Genet 4 e1000106 doi:10.1371/journal.pgen.1000106
63. AcarMPandoBFArnoldFHElowitzMBvan OudenaardenA 2010 A general mechanism of network-dosage compensation in gene circuits. Science 329 5999 1656 1660
64. AmbrosV 2004 The functions of animal microRNAs. Nature 431 350 355
65. BartelDP 2004 MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell 116 281 297
66. GaulU 2010 Decoding transcription and microRNA-mediated translation control in Drosophila development. Biol Chem 391 767 770
67. LiXCassidyJJReinkeCAFischboeckSCarthewRW 2009 A microRNA imparts robustness against environmental fluctuation during development. Cell 137 273 282
68. BoettigerANLevineM 2009 Synchronous and stochastic patterns of gene activation in the Drosophila embryo. Science 325 471 473
69. FrankelNDavisGKVargasDWang SPayreF Phenotypic robustness conferred by apparently redundant transcriptional enhancers. Nature 466 490 493
70. PerryMWBoettigerANBothmaJPLevineM 2010 Shadow enhancers foster robustness of Drosophila gastrulation. Curr Biol 20 1562 1567
71. PerryMWCandeJDBoettigerANLevineM 2009 Evolution of insect dorsoventral patterning mechanisms. Cold Spring Harb Symp Quant Biol 74 275 279
72. BarriereAGordonKLRuvinskyI 2011 Distinct functional constraints partition sequence conservation in a cis-regulatory element. PLoS Genet 7 e1002095 doi:10.1371/journal.pgen.1002095
73. ManuLudwigMZKreitmanM 2011 Sex-specific pattern formation during early Drosophila development. Proc Natl Acad Sci U S A In review
74. LottSEVillaltaJESchrothGPLuoSTonkinLA 2011 Noncanonical compensation of zygotic X transcription in early Drosophila melanogaster development revealed through single-embryo RNA-seq. PLoS Biol 9 e1000590 doi:10.1371/journal.pbio.1000590
75. RanzJMMaurinDChanYSvon GrotthussMHillierLW 2007 Principles of genome evolution in the Drosophila melanogaster species group. PLoS Biol 5 e152 doi:10.1371/journal.pbio.0050152
76. AndrioliLPMVasishtVTheodosopoulouEObersteinASmallS 2002 Anterior repression of a Drosophila stripe enhancer requires three position-specific mechanisms. Development 129 4931 4940
77. O'BrienMARobertsMSTaghertPH 1994 A genetic and molecular analysis of the 46C chromosomal region surrounding the FMRFamide neuropeptide gene in Drosophila melanogaster. Genetics 137 121 137
78. GoldsteinESTreadwaySLStephensonAEGramstadGDKeiltyA 2001 A genetic analysis of the cytological region 46C-F containing the Drosophila melanogaster homolog of the jun proto-oncogene. Mol Genet Genomics 266 695 700
79. GongWJGolicKG 2003 Ends-out, or replacement, gene targeting in Drosophila. Proc Natl Acad Sci U S A 100 2556 2561
80. GongWJGolicKG 2004 Genomic deletions of the Drosophila melanogaster Hsp70 genes. Genetics 168 1467 1476
81. RongYSGolicKG 2001 A targeted gene knockout in Drosophila. Genetics 157 1307 1312
82. RubinGMSpradlingAC 1982 Genetic transformation of Drosophila with transposable element vectors. Science 218 348 353
83. PatelNHCondronBGZinnK 1994 Pair-rule expression patterns of even-skipped are found in both short- and long-germ beetles. Nature 367 429 434
84. FoeVEAlbertsBM 1983 Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis. The Journal of Cell Science 61 31 70
85. JanssensHKosmanDVanario-AlonsoCEJaegerJSamsonovaM 2005 A high-throughput method for quantifying gene expression data from early Drosophila embryos. Dev Genes Evol 215 374 381
86. GonzalezRCWoodsRE 2002 Digital image processing. Upper Saddle River, NJ, USA Prentice-Hall Inc.
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
2011 Číslo 11
- Je „freeze-all“ pro všechny? Odborníci na fertilitu diskutovali na virtuálním summitu
- Gynekologové a odborníci na reprodukční medicínu se sejdou na prvním virtuálním summitu
Najčítanejšie v tomto čísle
- Evidence-Based Annotation of Gene Function in MR-1 Using Genome-Wide Fitness Profiling across 121 Conditions
- De Novo Origins of Human Genes
- TRY-5 Is a Sperm-Activating Protease in Seminal Fluid
- Cyclin D/CDK4 and Cyclin E/CDK2 Induce Distinct Cell Cycle Re-Entry Programs in Differentiated Muscle Cells