Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type–specific chromatin organization. However, factors regulating tissue-specific insulator function have not yet been discovered. Here we identify the RNA recognition motif-containing protein Shep as a direct interactor of two individual components of the gypsy insulator complex in Drosophila. Mutation of shep improves gypsy-dependent enhancer blocking, indicating a role as a negative regulator of insulator activity. Unlike ubiquitously expressed core gypsy insulator proteins, Shep is highly expressed in the central nervous system (CNS) with lower expression in other tissues. We developed a novel, quantitative tissue-specific barrier assay to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue. Additionally, mutation of shep alters insulator complex nuclear localization in the CNS but has no effect in other tissues. Consistent with negative regulatory activity, ChIP–seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single gypsy insulator component but limited overlap with intact insulator complexes. Taken together, these data reveal a novel, tissue-specific mode of regulation of a chromatin insulator.
Vyšlo v časopise: Tissue-Specific Regulation of Chromatin Insulator Function. PLoS Genet 8(11): e32767. doi:10.1371/journal.pgen.1003069 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003069
1. GasznerM, FelsenfeldG (2006) Insulators: exploiting transcriptional and epigenetic mechanisms. Nat Rev Genet 7 : 703–713.
2. PhillipsJE, CorcesVG (2009) CTCF: master weaver of the genome. Cell 137 : 1194–1211.
3. 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.
4. PaiCY, LeiEP, GhoshD, CorcesVG (2004) The centrosomal protein CP190 is a component of the gypsy chromatin insulator. Mol Cell 16 : 737–748.
5. GerasimovaTI, GdulaDA, GerasimovDV, SimonovaO, CorcesVG (1995) A Drosophila protein that imparts directionality on a chromatin insulator is an enhancer of position-effect variegation. Cell 82 : 587–597.
6. CaiHN, LevineM (1997) The gypsy insulator can function as a promoter-specific silencer in the Drosophila embryo. EMBO J 16 : 1732–1741.
7. GeyerPK, CorcesVG (1992) DNA position-specific repression of transcription by a Drosophila zinc finger protein. Genes Dev 6 : 1865–1873.
8. GerasimovaTI, ByrdK, CorcesVG (2000) A chromatin insulator determines the nuclear localization of DNA. Mol Cell 6 : 1025–1035.
9. BusheyAM, RamosE, CorcesVG (2009) Three subclasses of a Drosophila insulator show distinct and cell type-specific genomic distributions. Genes Dev 23 : 1338–1350.
10. NegreN, BrownCD, ShahPK, KheradpourP, MorrisonCA, et al. (2010) A comprehensive map of insulator elements for the Drosophila genome. PLoS Genet 6: e1000814 doi:10.1371/journal.pgen.1000814.
11. van BemmelJG, PagieL, BraunschweigU, BrugmanW, MeulemanW, et al. (2010) The insulator protein SU(HW) fine-tunes nuclear lamina interactions of the Drosophila genome. PLoS ONE 5: e15013 doi:10.1371/journal.pone.0015013.
12. GolovninA, VolkovI, GeorgievP (2012) SUMO conjugation is required for the assembly of Drosophila Su(Hw) and Mod(mdg4) into insulator bodies that facilitate insulator complex formation. J Cell Sci 125 : 2064–2074.
13. GerasimovaTI, CorcesVG (1998) Polycomb and trithorax group proteins mediate the function of a chromatin insulator. Cell 92 : 511–521.
14. GhoshD, GerasimovaTI, CorcesVG (2001) Interactions between the Su(Hw) and Mod(mdg4) proteins required for gypsy insulator function. EMBO J 20 : 2518–2527.
15. GolovninA, MelnikovaL, VolkovI, KostuchenkoM, GalkinAV, et al. (2008) ‘Insulator bodies’ are aggregates of proteins but not of insulators. EMBO Rep 9 : 440–445.
16. GerasimovaTI, LeiEP, BusheyAM, CorcesVG (2007) Coordinated control of dCTCF and gypsy chromatin insulators in Drosophila. Mol Cell 28 : 761–772.
17. CapelsonM, CorcesVG (2005) The ubiquitin ligase dTopors directs the nuclear organization of a chromatin insulator. Mol Cell 20 : 105–116.
18. RamosE, TorreEA, BusheyAM, GurudattaBV, CorcesVG (2011) DNA topoisomerase II modulates insulator function in Drosophila. PLoS ONE 6: e16562 doi:10.1371/journal.pone.0016562.
19. CapelsonM, CorcesVG (2006) SUMO conjugation attenuates the activity of the gypsy chromatin insulator. EMBO J 25 : 1906–1914.
20. LeiEP, CorcesVG (2006) RNA interference machinery influences the nuclear organization of a chromatin insulator. Nat Genet 38 : 936–941.
21. WoodAM, Van BortleK, RamosE, TakenakaN, RohrbaughM, et al. (2011) Regulation of chromatin organization and inducible gene expression by a Drosophila insulator. Mol Cell 44 : 29–38.
22. Lieberman-AidenE, van BerkumNL, WilliamsL, ImakaevM, RagoczyT, et al. (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326 : 289–293.
23. DixonJR, SelvarajS, YueF, KimA, LiY, et al. (2012) Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485 : 376–380.
24. HandokoL, XuH, LiG, NganCY, ChewE, et al. (2011) CTCF-mediated functional chromatin interactome in pluripotent cells. Nat Genet 43 : 630–638.
25. SextonT, YaffeE, KenigsbergE, BantigniesF, LeblancB, et al. (2012) Three-dimensional folding and functional organization principles of the Drosophila genome. Cell 148 : 458–472.
26. SplinterE, HeathH, KoorenJ, PalstraRJ, KlousP, et al. (2006) CTCF mediates long-range chromatin looping and local histone modification in the beta-globin locus. Genes Dev 20 : 2349–2354.
27. XuZ, WeiG, ChepelevI, ZhaoK, FelsenfeldG (2011) Mapping of INS promoter interactions reveals its role in long-range regulation of SYT8 transcription. Nat Struct Mol Biol 18 : 372–378.
28. SimonisM, KlousP, SplinterE, MoshkinY, WillemsenR, et al. (2006) Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C). Nat Genet 38 : 1348–1354.
29. HouC, DaleR, DeanA (2010) Cell type specificity of chromatin organization mediated by CTCF and cohesin. Proc Natl Acad Sci U S A 107 : 3651–3656.
30. LiHB, MullerM, BahecharIA, KyrchanovaO, OhnoK, et al. (2011) Insulators, not Polycomb response elements, are required for long-range interactions between Polycomb targets in Drosophila melanogaster. Mol Cell Biol 31 : 616–625.
31. WangKC, YangYW, LiuB, SanyalA, Corces-ZimmermanR, et al. (2011) A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472 : 120–124.
32. MoshkovichN, NishaP, BoylePJ, ThompsonBA, DaleRK, et al. (2011) RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function. Genes Dev 25 : 1686–1701.
33. ArmstrongJD, TexadaMJ, MunjaalR, BakerDA, BeckinghamKM (2006) Gravitaxis in Drosophila melanogaster: a forward genetic screen. Genes Brain Behav 5 : 222–239.
34. BellenHJ, LevisRW, LiaoG, HeY, CarlsonJW, et al. (2004) The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics 167 : 761–781.
35. RyderE, BlowsF, AshburnerM, Bautista-LlacerR, CoulsonD, et al. (2004) The DrosDel collection: a set of P-element insertions for generating custom chromosomal aberrations in Drosophila melanogaster. Genetics 167 : 797–813.
36. GdulaDA, GerasimovaTI, CorcesVG (1996) Genetic and molecular analysis of the gypsy chromatin insulator of Drosophila. Proc Natl Acad Sci U S A 93 : 9378–9383.
37. ChintapalliVR, WangJ, DowJA (2007) Using FlyAtlas to identify better Drosophila melanogaster models of human disease. Nat Genet 39 : 715–720.
38. MarksteinM, PitsouliC, VillaltaC, CelnikerSE, PerrimonN (2008) Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nat Genet 40 : 476–483.
39. DietzlG, ChenD, SchnorrerF, SuKC, BarinovaY, et al. (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448 : 151–156.
40. GrothAC, FishM, NusseR, CalosMP (2004) Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics 166 : 1775–1782.
41. MoshkovichN, LeiEP (2010) HP1 recruitment in the absence of argonaute proteins in Drosophila. PLoS Genet 6: e1000880 doi:10.1371/journal.pgen.1000880.
42. Van BortleK, RamosE, TakenakaN, YangJ, WahiJE, et al. (2012) Drosophila CTCF tandemly aligns with other insulator proteins at the borders of H3K27me3 domains. Genome Res In press.
43. KharchenkoPV, TolstorukovMY, ParkPJ (2008) Design and analysis of ChIP-seq experiments for DNA-binding proteins. Nat Biotechnol 26 : 1351–1359.
44. SchwartzYB, Linder-BassoD, KharchenkoPV, TolstorukovMY, KimM, et al. (2012) Nature and function of insulator protein binding sites in the Drosophila genome. Genome Res In press.
45. KimTH, AbdullaevZK, SmithAD, ChingKA, LoukinovDI, et al. (2007) Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome. Cell 128 : 1231–1245.
46. EdwardsAC, ZwartsL, YamamotoA, CallaertsP, MackayTF (2009) Mutations in many genes affect aggressive behavior in Drosophila melanogaster. BMC Biol 7 : 29.
47. MongelardF, LabradorM, BaxterEM, GerasimovaTI, CorcesVG (2002) Trans-splicing as a novel mechanism to explain interallelic complementation in Drosophila. Genetics 160 : 1481–1487.
48. KahnTG, SchwartzYB, DellinoGI, PirrottaV (2006) Polycomb complexes and the propagation of the methylation mark at the Drosophila ubx gene. J Biol Chem 281 : 29064–29075.
49. AmeroSA, ElginSC, BeyerAL (1991) A unique zinc finger protein is associated preferentially with active ecdysone-responsive loci in Drosophila. Genes Dev 5 : 188–200.
50. DeFalcoTJ, VerneyG, JenkinsAB, McCafferyJM, RussellS, et al. (2003) Sex-specific apoptosis regulates sexual dimorphism in the Drosophila embryonic gonad. Dev Cell 5 : 205–216.
51. PatelNH (1994) Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes. Methods Cell Biol 44 : 445–487.
52. DaleRK, PedersenBS, QuinlanAR (2011) Pybedtools: a flexible Python library for manipulating genomic datasets and annotations. Bioinformatics 27 : 3423–3424.
53. QuinlanAR, HallIM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26 : 841–842.
54. CelnikerSE, DillonLA, GersteinMB, GunsalusKC, HenikoffS, et al. (2009) Unlocking the secrets of the genome. Nature 459 : 927–930.
55. SchwartzYB, KahnTG, StenbergP, OhnoK, BourgonR, et al. (2010) Alternative epigenetic chromatin states of polycomb target genes. PLoS Genet 6: e1000805 doi:10.1371/journal.pgen.1000805.
56. RichterC, OktabaK, SteinmannJ, MullerJ, KnoblichJA (2011) The tumour suppressor L(3)mbt inhibits neuroepithelial proliferation and acts on insulator elements. Nat Cell Biol 13 : 1029–1039.
57. FavorovA, MularoniL, CopeLM, MedvedevaY, MironovAA, et al. (2012) Exploring massive, genome scale datasets with the GenometriCorr package. PLoS Comput Biol 8: e1002529 doi:10.1371/journal.pcbi.1002529.
58. SchuettengruberB, GanapathiM, LeblancB, PortosoM, JaschekR, et al. (2009) Functional anatomy of polycomb and trithorax chromatin landscapes in Drosophila embryos. PLoS Biol 7: e13 doi:10.1371/journal.pbio.1000013.
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