Effective Blocking of the Enhancer Requires Cooperation between Two Main Mechanisms Suggested for the Insulator Function

English version České info

Chromatin insulators block the action of transcriptional enhancers when interposed between an enhancer and a promoter. In this study, we examined the role of chromatin loops formed by two unrelated insulators, gypsy and Fab-7, in their enhancer-blocking activity. To test for this activity, we selected the white reporter gene that is activated by the eye-specific enhancer. The results showed that one copy of the gypsy or Fab-7 insulator failed to block the eye enhancer in most of genomic sites, whereas a chromatin loop formed by two gypsy insulators flanking either the eye enhancer or the reporter completely blocked white stimulation by the enhancer. However, strong enhancer blocking was achieved due not only to chromatin loop formation but also to the direct interaction of the gypsy insulator with the eye enhancer, which was confirmed by the 3C assay. In particular, it was observed that Mod(mdg4)-67.2, a component of the gypsy insulator, interacted with the Zeste protein, which is critical for the eye enhancer–white promoter communication. These results suggest that efficient enhancer blocking depends on the combination of two factors: chromatin loop formation by paired insulators, which generates physical constraints for enhancer–promoter communication, and the direct interaction of proteins recruited to an insulator and to the enhancer–promoter pair.

Vyšlo v časopise: Effective Blocking of the Enhancer Requires Cooperation between Two Main Mechanisms Suggested for the Insulator Function. PLoS Genet 9(7): e32767. doi:10.1371/journal.pgen.1003606
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003606

Souhrn

Zdroje

1. Kuhn EJ, GeyerPK (2003) Genomic insulators: Connecting properties to mechanism. Curr Opin Cell Biol 15 : 259–265.

2. BrassetE, VauryC (2005) Insulators are fundamental components of the eukaryotic genomes. Heredity 94(6): 571–576.

3. ZhaoH, DeanA (2005) Organizing the genome: Enhancers and insulators. Biochem Cell Biol 83 : 516–524.

4. WallaceJA, FelsenfeldG (2007) We gather together: Insulators and genome organization. Curr Opin Genet Dev 17 : 400–407.

5. ValenzuelaL, KamakakaRT (2006) Chromatin insulators. Annu Rev Genet 40 : 107–138.

6. MaksimenkoOG, ChetverinaDA, GeorgievPG (2006) Insulators of higher eukaryotes: Properties, mechanisms of action, and role in transcription regulation. Russ J Genet 42 : 845–857.

7. BarkessG, WestAG (2012) Chromatin insulator elements: Establishing barriers to set heterochromatin boundaries. Epigenomics 4 : 67–80.

8. KyrchanovaO, ChetverinaD, MaksimenkoO, KullyevA, GeorgievP (2008) Orientation-dependent interaction between Drosophila insulators is a property of this class of regulatory elements. Nucleic Acids Res 36 : 7019–7028.

9. MaksimenkoO, GolovninA, GeorgievP (2008) Enhancer-promoter communication is regulated by insulator pairing in a Drosophila model bigenic locus. Mol Cell Biol 28 : 5469–5477.

10. ChetverinaD, SavitskayaE, MaksimenkoO, MelnikovaL, ZaytsevaO (2008) Red flag on the white reporter: A versatile insulator abuts the white gene in Drosophila and is omnipresent in mini-white constructs. Nucleic Acids Res 36 : 929–37.

11. KravchenkoE, SavitskayaE, KravchukO, ParshikovA, GeorgievP, et al. (2005) Pairing between gypsy insulators facilitates the enhancer action in trans throughout the Drosophila genome. Mol Cell Biol 25 : 9283–9291.

12. LiHB, MullerM, BahecharIA, KyrchanovaO, OhnoK, et al. (2011) Insulators, not Polycomb Response Elements, are required for long-distance interactions between Polycomb targets in Drosophila melanogaster. Mol Cell Biol 31 : 616–625.

13. AdryanB, WoerfelG, Birch-MachinI, GaoS, QuickM, et al. (2007) Genomic mapping of Suppressor of Hairy-wing binding sites in Drosophila. Genome Biol 8: R167.

14. NègreN, BrownCD, ShahPK, KheradpourP, MorrisonCA, et al. (2010) A comprehensive map of insulator elements for the Drosophila genome. PloS Genet 6: e1000814.

15. NègreN, BrownCD, MaL, BristowCA, MillerS, et al. (2011) A cis-regulatory map of the Drosophila genome. Nature 471 : 527–531.

16. BartkuhnM, StaubT, HeroldM, HerrmannM, RathkeC, et al. (2009) Active promoters and insulators are marked by the centrosomal protein 190. EMBO J 28 : 877–888.

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

18. KehayovaP, MonahanK, ChenW, ManiatisT (2011) Regulatory elements required for the activation and repression of the protocadherin-{alpha} gene cluster. Proc Natl Acad Sci USA 108 : 17195–17200.

19. LiuZ, ScannellDR, EisenMB, TjianR (2011) Control of embryonic stem cell lineage commitment by core promoter factor, TAF3. Cell 146 : 720–731.

20. HandokoL, XuH, LiG, NganCY, ChewE, et al. (2011) CTCF-mediated functional chromatin interactome in pluripotent cells. Nature Genet 43 : 630–638.

21. DixonJR, SelvarajS, YueF, KimA, LiY, et al. (2012) Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485 : 376–380.

22. SextonT, YaffeE, KenigsbergE, BantigniesF, LeblancB, et al. (2012) Three-dimensional folding and functional organization principles of the Drosophila genome. Cell 148 : 458–472.

23. KellumR, SchedlP (1991) A position-effect assay for boundaries of higher order chromosomal domains. Cell 64 : 941–950.

24. KellumR, SchedlP (1992) A group of scs elements function as domain boundaries in an enhancer-blocking assay. Mol Cell Biol 12 : 2424–2431.

25. HagstromK, MullerM, SchedlP (1996) Fab-7 functions as a chromatin domain boundary to ensure proper segment specification by the Drosophila bithorax complex. Genes Dev 10 : 3202–3215.

26. ZhouJ, BaroloS, SzymanskiP, LevineM (1996) The Fab-7 element of the bithorax complex attenuates enhancer-promoter interactions in the Drosophila embryo. Genes Dev 10 : 3195–3201.

27. BargesS, MihalyJ, GalloniM, HagstromK, MullerM, et al. (2000) The Fab-8 boundary defines the distal limit of the bithorax complex iab-7 domain and insulates iab-7 from initiation elements and a PRE in the adjacent iab-8 domain. Development 127 : 779–790.

28. SchweinsbergS, SchedlP (2004) Developmental modulation of Fab-7 boundary function. Development 131 : 4743–4749.

29. GruzdevaN, KyrchanovaO, ParshikovA, KullyevA, GeorgievP (2005) The Mcp element from the bithorax complex contains an insulator that is capable of pairwise interactions and can facilitate enhancer–promoter communication. Mol Cell Biol 25 : 3682–3689.

30. BelozerovVE, MajumderP, ShenP, CaiHN (2003) A novel boundary element may facilitate independent gene regulation in the Antennapedia complex of Drosophila. EMBO J 22 : 3113–3121.

31. ConteC, DastugueB, VauryC (2002) Coupling of enhancer and insulator properties identified in two retrotransposons modulates their mutagenic impact on nearby genes. Mol Cell Biol 22 : 1767–1777.

32. HoldridgeC, DorsettD (1991) Repression of hsp70 heat shock gene transcription by the suppressor of hairy-wing protein of Drosophila melanogaster. Mol Cell Biol 11 : 1894–1900.

33. GeyerPK, CorcesVG (1992) DNA position-specific repression of transcription by a Drosophila zinc finger protein. Genes Dev 6 : 1865–1873.

34. GolovninA, BiryukovaI, RomanovaO, SilichevaM, ParshikovA, et al. (2003) An endogenous Su(Hw) insulator separates the yellow gene from the Achaete-scute gene complex in Drosophila. Development 130 : 3249–3258.

35. ParnellTJ, VieringMM, SkjesolA, HelouC, KuhnEJ, et al. (2003) An endogenous suppressor of hairy-wing insulator separates regulatory domains in Drosophila. Proc Natl Acad Sci U S A 100 : 13436–13441.

36. ParnellTJ, KuhnEJ, GilmoreBL, HelouC, WoldMS, et al. (2006) Identification of genomic sites that bind the Drosophila suppressor of Hairy-wing insulator protein. Mol Cell Biol 26 : 5983–5993.

37. HeroldM, BartkuhnM, RenkawitzR (2012) CTCF: Insights into insulator function during development. Development 139 : 1045–1057.

38. CaiHN, ShenP (2001) Effects of cis arrangement of chromatin insulators on enhancer-blocking activity. Science 291 : 493–495.

39. MuravyovaE, GolovninA, GrachevaE, ParshikovA, BelenkayaT, et al. (2001) Loss of insulator activity by paired Su(Hw) chromatin insulators. Science 291 : 495–498.

40. BlantonJ, GasznerM, SchedlP (2003) Protein : protein interactions and the pairing of boundary elements in vivo. Genes Dev 17 : 664–675.

41. AmeresSL, DrueppelL, PfleidererK, SchmidtA, HillenW, et al. (2005) Inducible DNA-loop formation blocks transcriptional activation by an SV40 enhancer. EMBO J 24 : 358–367.

42. BondarenkoVA, LiuYV, JiangYI, StuditskyVM (2003) Communication over a large distance: Enhancers and insulators. Biochem Cell Biol 81 : 241–251.

43. BondarenkoVA, JiangYI, StuditskyVM (2003) Rationally designed insulator-like elements can block enhancer action in vitro. EMBO J 22 : 4728–4737.

44. MukhopadhyayS, SchedlP, StuditskyVM, SenguptaAM (2011) Theoretical analysis of the role of chromatin interactions in long-range action of enhancers and insulators. Proc Natl Acad Sci U S A 108 : 19919–19924.

45. GohlD, AokiT, BlantonJ, ShanowerG, KappesG, et al. (2011) Mechanism of chromosomal boundary action: Roadblock, sink, or loop? Genetics 187 : 731–748.

46. HouC, ZhaoH, TanimotoK, DeanA (2008) CTCF-dependent enhancer-blocking by alternative chromatin loop formation. Proc Natl Acad Sci U S A 105 : 20398–20403.

47. GauseM, MorcilloP, DorsettD (2001) Insulation of enhancer–promoter communication by a gypsy transposon insert in the Drosophila cut gene: Cooperation between suppressor of hairy-wing and modifier of mdg4 proteins. Mol Cell Biol 21 : 4807–4817.

48. GhoshD, GerasimovaTI, CorcesVG (2001) Interactions between the Su(Hw) and Mod(mdg4) proteins required for gypsy insulator function. EMBO J 20 : 2518–2527.

49. PaiC-Y, LeiEP, GhoshD, CorcesVG (2004) The centrosomal protein CP190 is a component of the gypsy chromatin insulator. Mol Cell 16 : 737–748.

50. KurshakovaM, MaksimenkoO, GolovninA, PulinaM, GeorgievaS, et al. (2007) Evolutionarily conserved E(y)2/Sus1 protein is essential for the barrier activity of Su(Hw)-dependent insulators in Drosophila. Mol Cell 27 : 332–338.

51. GolovninA, MazurA, KopantsevaM, KurshakovaM, GulakPV, et al. (2007) Integrity of the Mod(mdg4)-67.2 BTB domain is critical to insulator function in Drosophila. Mol Cell Biol 27 : 963–974.

52. BonchukA, DenisovS, GeorgievP, MaksimenkoO (2011) Drosophila BTB/Poz domains of “ttk group” can form multimers and selectively interact with each other. J Mol Biol 412 : 423–436.

53. KrivegaM, SavitskayaE, KrivegaI, KarakozovaM, ParshikovA, et al. (2010) Interaction between a pair of gypsy insulators or between gypsy and Wari insulators modulates Flp site-specific recombination in Drosophila melanogaster. Chromosoma 119 : 425–434.

54. CometI, SchuettengruberB, SextonT, CavalliG (2011) A chromatin insulator driving three-dimensional Polycomb response element (PRE) contacts and Polycomb association with the chromatin fiber. Proc Natl Acad Sci U S A 108 : 2294–2299.

55. MaedaRK, KarchF (2011) Gene expression in time and space: Additive vs. hierarchical organization of cis-regulatory regions. Curr Opin Genet Dev 21 : 187–193.

56. GyurkovicsH, GauszJ, KummerJ, KarchF (1990) A new homeotic mutation in the Drosophila bithorax complex removes a boundary separating two domains of regulation. EMBO J 9 : 2579–2585.

57. GalloniM, GyurkovicsH, SchedlP, KarchF (1993) The bluetail transposon: Evidence for independent cis-regulatory domains and domain boundaries in the bithorax complex. EMBO J 12 : 1087–1097.

58. KarchF, GalloniM, SiposL, GauszJ, GyurkovicsH, et al. (1994) Mcp and Fab-7: Molecular analysis of putative boundaries of cis-regulatory domains in the bithorax complex of Drosophila melanogaster. Nucleic Acids Res 22 : 3138–3146.

59. RodinS, KyrchanovaO, PomerantsevaE, ParshikovA, GeorgievP (2007) New properties of Drosophila Fab-7 insulator. Genetics 177 : 113–121.

60. KuhnEJ, VieringMM, RhodesKM, GeyerPK (2003) A test of insulator interactions in Drosophila. EMBO J 22 : 2463–2471.

61. SchweinsbergS, HagstromK, GohlD, SchedlP, KumarRP, et al. (2004) The enhancer-blocking activity of the Fab-7 boundary from the Drosophila bithorax complex requires GAGA-factor-binding sites. Genetics 168 : 1371–1384.

62. Savitskaya E, Melnikova L, Kostuchenko M, Kravchenko E, Pomerantseva E, et al. 2006. Study of long-distance functional interactions between Su(Hw) insulators that can regulate enhancer–promoter communication in Drosophila melanogaster. Mol Cell Biol 26 : 754–761.

63. MelnikovaL, KostuchenkoM, SilichevaM, GeorgievP (2008) Drosophila gypsy insulator and yellow enhancers regulate activity of yellow promoter through the same regulatory element. Chromosoma 117 : 137–145.

64. QianS, VarjavandB, PirrottaV (1992) Molecular analysis of the zeste-white interaction reveals a promoter-proximal element essential for distant enhancer-promoter communication. Genetics 131 : 79–90.

65. PirrottaV, ManetE, HardonE, BickelSE, BensonM (1987) Structure and sequence of the Drosophila zeste gene. EMBO J 6 : 791–799.

66. HarrisonDA, GdulaDA, CoyneRS, CorcesVG (1993) A leucine zipper domain of the suppressor of Hairy-wing protein mediates its repressive effect on enhancer function. Genes Dev 7 : 1966–1978.

67. KimJ, ShenB, RosenC, DorsettD (1996) The DNA-binding and enhancer-blocking domains of the Drosophila suppressor of Hairy-wing protein. Mol Cell Biol 16 : 3381–3392.

68. GeorgievP, KozycinaM (1996) Interaction between mutations in the suppressor of Hairy wing and modifier of mdg4 genes of Drosophila melanogaster affecting the phenotype of gypsy-induced mutations. Genetics 142 : 425–436.

69. CaiHN, LevineM (1997) The gypsy insulator can function as a promoter-specific silencer in the Drosophila embryo. EMBO J 16 : 1732–1741.

70. CapelsonM, CorcesVG (2005) The ubiquitin ligase dTopors directs the nuclear organization of a chromatin insulator. Mol Cell 20 : 105–116.

71. KostyuchenkoM, SavitskayaE, KoryaginaE, MelnikovaL, KarakozovaM, et al. (2009) Zeste can facilitate long-range enhancer–promoter communication and insulator bypass in Drosophila melanogaster. Chromosoma 118 : 665–674.

72. RaabJR, KamakakaRT (2010) Insulators and promoters: Closer than we think. Nature Rev Genet 11 : 439–446.

73. ChopraVS, CandeJ, HongJW, LevineM (2009) Stalled Hox promoters as chromosomal boundaries. Genes Dev 23 : 1505–1509.

74. ErokhinM, DavydovaA, KyrchanovaO, ParshikovA, GeorgievP, et al. (2011) Insulators form gene loops by interacting with promoters in Drosophila. Development 138 : 4097–4106.

75. KaressRE, RubinGM (1984) Analysis of P transposable element functions in Drosophila. Cell 38 : 135–146.

76. GolicKG, LindquistS (1989) The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59 : 499–509.

77. SiegalML, HartlDL (2000) Application of Cre/loxP in Drosophila: Site-specific recombination and transgene coplacement. Methods Mol Biol 136 : 487–495.

78. PirrottaV (1988) Vectors for P-mediated transformation in Drosophila. Biotechnology 10 : 437–456.

79. BensonM, PirrottaV (1987) The product of the Drosophila zeste gene binds to specific DNA sequences in white and Ubx. EMBO J 6 : 1387–1392.

80. HagègeH, KlousP, BraemC, SplinterE, DekkerJ, CathalaG, de LaatW, FornéT (2007) Quantitative analysis of chromosome conformation capture assays (3C-qPCR). Nature Protoc 2 : 1722–1733.

81. MoshkovichN, NishaP, BoylePJ, ThompsonBA, DaleRK, LeiEP (2011) RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function. Genes Dev 25 : 1686–1701.