#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Downregulation and Altered Splicing by in a Mouse Model of Facioscapulohumeral Muscular Dystrophy (FSHD)


Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1) in mice, frogs, and worms perturbs muscle development and causes FSHD–like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6–) is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.


Vyšlo v časopise: Downregulation and Altered Splicing by in a Mouse Model of Facioscapulohumeral Muscular Dystrophy (FSHD). PLoS Genet 9(1): e32767. doi:10.1371/journal.pgen.1003186
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003186

Souhrn

Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1) in mice, frogs, and worms perturbs muscle development and causes FSHD–like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6–) is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.


Zdroje

1. CabiancaDS, GabelliniD (2010) The cell biology of disease: FSHD: copy number variations on the theme of muscular dystrophy. J Cell Biol 191: 1049–1060.

2. FlaniganKM, CoffeenCM, SextonL, StaufferD, BrunnerS, et al. (2001) Genetic characterization of a large, historically significant Utah kindred with facioscapulohumeral dystrophy. Neuromuscul Disord 11: 525–529.

3. PandyaS, KingWM, TawilR (2008) Facioscapulohumeral dystrophy. Phys Ther 88: 105–113.

4. ShahrizailaN, WillsAJ (2005) Significance of Beevor's sign in facioscapulohumeral dystrophy and other neuromuscular diseases. J Neurol Neurosurg Psychiatry 76: 869–870.

5. BarroM, CarnacG, FlavierS, MercierJ, VassetzkyY, et al. (2008) Myoblasts from affected and non-affected FSHD muscles exhibit morphological differentiation defects. J Cell Mol Med 14: 275–289.

6. CelegatoB, CapitanioD, PescatoriM, RomualdiC, PacchioniB, et al. (2006) Parallel protein and transcript profiles of FSHD patient muscles correlate to the D4Z4 arrangement and reveal a common impairment of slow to fast fibre differentiation and a general deregulation of MyoD-dependent genes. Proteomics 6: 5303–5321.

7. MorosettiR, MirabellaM, GliubizziC, BroccoliniA, SancriccaC, et al. (2007) Isolation and characterization of mesoangioblasts from facioscapulohumeral muscular dystrophy muscle biopsies. Stem Cells 25: 3173–3182.

8. StadlerG, ChenJC, WagnerK, RobinJD, ShayJW, et al. (2011) Establishment of clonal myogenic cell lines from severely affected dystrophic muscles - CDK4 maintains the myogenic population. Skelet Muscle 1: 12.

9. TuplerR, PeriniG, PellegrinoMA, GreenMR (1999) Profound misregulation of muscle-specific gene expression in facioscapulohumeral muscular dystrophy. Proc Natl Acad Sci U S A 96: 12650–12654.

10. WinokurST, BarrettK, MartinJH, ForresterJR, SimonM, et al. (2003) Facioscapulohumeral muscular dystrophy (FSHD) myoblasts demonstrate increased susceptibility to oxidative stress. Neuromuscul Disord 13: 322–333.

11. WinokurST, ChenYW, MasnyPS, MartinJH, EhmsenJT, et al. (2003) Expression profiling of FSHD muscle supports a defect in specific stages of myogenic differentiation. Hum Mol Genet 12: 2895–2907.

12. BrouwerOF, PadbergGW, BakkerE, WijmengaC, FrantsRR (1995) Early onset facioscapulohumeral muscular dystrophy. Muscle Nerve 2: S67–72.

13. FunakoshiM, GotoK, ArahataK (1998) Epilepsy and mental retardation in a subset of early onset 4q35-facioscapulohumeral muscular dystrophy. Neurology 50: 1791–1794.

14. MiuraK, KumagaiT, MatsumotoA, IriyamaE, WatanabeK, et al. (1998) Two cases of chromosome 4q35-linked early onset facioscapulohumeral muscular dystrophy with mental retardation and epilepsy. Neuropediatrics 29: 239–241.

15. SaitoY, MiyashitaS, YokoyamaA, KomakiH, SekiA, et al. (2007) Facioscapulohumeral muscular dystrophy with severe mental retardation and epilepsy. Brain Dev 29: 231–233.

16. NeguemborM, GabelliniD (2010) In Junk We Trust: Repetitive DNA, Epigenetics and Facioscapulohumeral Muscular Dystrophy. Epigenomics 2: 271–287.

17. HewittJE, LyleR, ClarkLN, ValleleyEM, WrightTJ, et al. (1994) Analysis of the tandem repeat locus D4Z4 associated with facioscapulohumeral muscular dystrophy. Hum Mol Genet 3: 1287–1295.

18. van DeutekomJC, WijmengaC, van TienhovenEA, GruterAM, HewittJE, et al. (1993) FSHD associated DNA rearrangements are due to deletions of integral copies of a 3.2 kb tandemly repeated unit. Hum Mol Genet 2: 2037–2042.

19. WijmengaC, HewittJE, SandkuijlLA, ClarkLN, WrightTJ, et al. (1992) Chromosome 4q DNA rearrangements associated with facioscapulohumeral muscular dystrophy. Nat Genet 2: 26–30.

20. WinokurST, BengtssonU, FeddersenJ, MathewsKD, WeiffenbachB, et al. (1994) The DNA rearrangement associated with facioscapulohumeral muscular dystrophy involves a heterochromatin-associated repetitive element: implications for a role of chromatin structure in the pathogenesis of the disease. Chromosome Res 2: 225–234.

21. CabiancaDS, CasaV, BodegaB, XynosA, GinelliE, et al. (2012) A long ncRNA links copy number variation to a polycomb/trithorax epigenetic switch in FSHD muscular dystrophy. Cell 149: 819–831.

22. GabelliniD, GreenMR, TuplerR (2002) Inappropriate gene activation in FSHD: a repressor complex binds a chromosomal repeat deleted in dystrophic muscle. Cell 110: 339–348.

23. LemmersRJ, van der VlietPJ, KloosterR, SacconiS, CamanoP, et al. (2010) A unifying genetic model for facioscapulohumeral muscular dystrophy. Science 329: 1650–1653.

24. HanelML, WuebblesRD, JonesPL (2009) Muscular dystrophy candidate gene FRG1 is critical for muscle development. Dev Dyn 238: 1502–1512.

25. GabelliniD, D'AntonaG, MoggioM, PrelleA, ZeccaC, et al. (2006) Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1. Nature 439: 973–977.

26. LiuQ, JonesTI, TangVW, BrieherWM, JonesPL (2010) Facioscapulohumeral muscular dystrophy region gene-1 (FRG-1) is an actin-bundling protein associated with muscle-attachment sites. J Cell Sci 123: 1116–1123.

27. WuebblesRD, HanelML, JonesPL (2009) FSHD region gene 1 (FRG1) is crucial for angiogenesis linking FRG1 to facioscapulohumeral muscular dystrophy-associated vasculopathy. Dis Model Mech 2: 267–274.

28. BessonovS, AnokhinaM, KrasauskasA, GolasMM, SanderB, et al. (2010) Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis. Rna 16: 2384–2403.

29. DavidovicL, SacconiS, BecharaEG, DelplaceS, AllegraM, et al. (2008) Alteration of expression of muscle specific isoforms of the fragile X related protein 1 (FXR1P) in facioscapulohumeral muscular dystrophy patients. J Med Genet 45: 679–685.

30. JuricaMS, LickliderLJ, GygiSR, GrigorieffN, MooreMJ (2002) Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis. Rna 8: 426–439.

31. KimSK, LundJ, KiralyM, DukeK, JiangM, et al. (2001) A gene expression map for Caenorhabditis elegans. Science 293: 2087–2092.

32. MakarovEM, MakarovaOV, UrlaubH, GentzelM, WillCL, et al. (2002) Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome. Science 298: 2205–2208.

33. RappsilberJ, RyderU, LamondAI, MannM (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res 12: 1231–1245.

34. van KoningsbruggenS, DirksRW, MommaasAM, OnderwaterJJ, DeiddaG, et al. (2004) FRG1P is localised in the nucleolus, Cajal bodies, and speckles. J Med Genet 41: e46.

35. KuroyanagiH (2009) Fox-1 family of RNA-binding proteins. Cell Mol Life Sci 66: 3895–3907.

36. DamianovA, BlackDL (2009) Autoregulation of Fox protein expression to produce dominant negative splicing factors. Rna 16: 405–416.

37. McKeeAE, MinetE, SternC, RiahiS, StilesCD, et al. (2005) A genome-wide in situ hybridization map of RNA-binding proteins reveals anatomically restricted expression in the developing mouse brain. BMC Dev Biol 5: 14.

38. TangZZ, ZhengS, NikolicJ, BlackDL (2009) Developmental control of CaV1.2 L-type calcium channel splicing by Fox proteins. Mol Cell Biol 29: 4757–4765.

39. BaraniakAP, ChenJR, Garcia-BlancoMA (2006) Fox-2 mediates epithelial cell-specific fibroblast growth factor receptor 2 exon choice. Mol Cell Biol 26: 1209–1222.

40. PonthierJL, SchluepenC, ChenW, LerschRA, GeeSL, et al. (2006) Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16. J Biol Chem 281: 12468–12474.

41. UnderwoodJG, BoutzPL, DoughertyJD, StoilovP, BlackDL (2005) Homologues of the Caenorhabditis elegans Fox-1 protein are neuronal splicing regulators in mammals. Mol Cell Biol 25: 10005–10016.

42. YeoGW, CoufalNG, LiangTY, PengGE, FuXD, et al. (2009) An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat Struct Mol Biol 16: 130–137.

43. KimKK, AdelsteinRS, KawamotoS (2009) Identification of neuronal nuclei (NeuN) as Fox-3, a new member of the Fox-1 gene family of splicing factors. J Biol Chem 284: 31052–31061.

44. NiJZ, GrateL, DonohueJP, PrestonC, NobidaN, et al. (2007) Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay. Genes Dev 21: 708–718.

45. SugnetCW, SrinivasanK, ClarkTA, O'BrienG, ClineMS, et al. (2006) Unusual intron conservation near tissue-regulated exons found by splicing microarrays. PLoS Comput Biol 2: e4 doi:10.1371/journal.pcbi.0020004.

46. WalshFS, CelesteAJ (2005) Myostatin: a modulator of skeletal-muscle stem cells. Biochem Soc Trans 33: 1513–1517.

47. BerkesCA, TapscottSJ (2005) MyoD and the transcriptional control of myogenesis. Semin Cell Dev Biol 16: 585–595.

48. Finanger HedderickEL, SimmersJL, SoleimaniA, Andres-MateosE, MarxR, et al. (2011) Loss of sarcolemmal nNOS is common in acquired and inherited neuromuscular disorders. Neurology 76: 960–967.

49. RichardI, BrouxO, AllamandV, FougerousseF, ChiannilkulchaiN, et al. (1995) Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A. Cell 81: 27–40.

50. SorimachiH, Imajoh-OhmiS, EmoriY, KawasakiH, OhnoS, et al. (1989) Molecular cloning of a novel mammalian calcium-dependent protease distinct from both m- and mu-types. Specific expression of the mRNA in skeletal muscle. J Biol Chem 264: 20106–20111.

51. BulfieldG, SillerWG, WightPA, MooreKJ (1984) X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci U S A 81: 1189–1192.

52. SunCY, van KoningsbruggenS, LongSW, StraasheijmK, KloosterR, et al. (2011) Facioscapulohumeral muscular dystrophy region gene 1 is a dynamic RNA-associated and actin-bundling protein. J Mol Biol 411: 397–416.

53. YamashitaR, SathiraNP, KanaiA, TanimotoK, ArauchiT, et al. (2011) Genome-wide characterization of transcriptional start sites in humans by integrative transcriptome analysis. Genome Res 21: 775–789.

54. SpencerMJ, GuyonJR, SorimachiH, PottsA, RichardI, et al. (2002) Stable expression of calpain 3 from a muscle transgene in vivo: immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation. Proc Natl Acad Sci U S A 99: 8874–8879.

55. SniderL, AsawachaicharnA, TylerAE, GengLN, PetekLM, et al. (2009) RNA transcripts, miRNA-sized fragments and proteins produced from D4Z4 units: new candidates for the pathophysiology of facioscapulohumeral dystrophy. Hum Mol Genet 18: 2414–2430.

56. SniderL, GengLN, LemmersRJ, KybaM, WareCB, et al. (2010) Facioscapulohumeral dystrophy: incomplete suppression of a retrotransposed gene. PLoS Genet 6: e1001181 doi:10.1371/journal.pgen.1001181.

57. WallaceLM, GarwickSE, MeiW, BelayewA, CoppeeF, et al. (2011) DUX4, a candidate gene for facioscapulohumeral muscular dystrophy, causes p53-dependent myopathy in vivo. Ann Neurol 69: 540–552.

58. BosnakovskiD, XuZ, GangEJ, GalindoCL, LiuM, et al. (2008) An isogenetic myoblast expression screen identifies DUX4-mediated FSHD-associated molecular pathologies. EMBO J 27: 2766–2779.

59. ArahataK, IshiharaT, FukunagaH, OrimoS, LeeJH, et al. (1995) Inflammatory response in facioscapulohumeral muscular dystrophy (FSHD): immunocytochemical and genetic analyses. Muscle Nerve 2: S56–66.

60. Figarella-BrangerD, PellissierJF, SerratriceG, PougetJ, BiancoN (1989) [Immunocytochemical study of the inflammatory forms of facioscapulohumeral myopathies and correlation with other types of myositis]. Ann Pathol 9: 100–108.

61. FrisulloG, FruscianteR, NocitiV, TascaG, RennaR, et al. (2011) CD8(+) T cells in facioscapulohumeral muscular dystrophy patients with inflammatory features at muscle MRI. J Clin Immunol 31: 155–166.

62. MunsatTL, PiperD, CancillaP, MednickJ (1972) Inflammatory myopathy with facioscapulohumeral distribution. Neurology 22: 335–347.

63. GengLN, YaoZ, SniderL, FongAP, CechJN, et al. (2012) DUX4 activates germline genes, retroelements, and immune mediators: implications for facioscapulohumeral dystrophy. Dev Cell 22: 38–51.

64. BhallaK, PhillipsHA, CrawfordJ, McKenzieOL, MulleyJC, et al. (2004) The de novo chromosome 16 translocations of two patients with abnormal phenotypes (mental retardation and epilepsy) disrupt the A2BP1 gene. J Hum Genet 49: 308–311.

65. DavisLK, MaltmanN, MosconiMW, MacmillanC, SchmittL, et al. (2012) Rare inherited A2BP1 deletion in a proband with autism and developmental hemiparesis. Am J Med Genet A 158A: 1654–1661.

66. FogelBL, WexlerE, WahnichA, FriedrichT, VijayendranC, et al. (2012) RBFOX1 regulates both splicing and transcriptional networks in human neuronal development. Hum Mol Genet 21: 4171–4186.

67. GallantNM, BaldwinE, SalamonN, DippleKM, Quintero-RiveraF (2011) Pontocerebellar hypoplasia in association with de novo 19p13.11p13.12 microdeletion. Am J Med Genet A 155A: 2871–2878.

68. GehmanLT, StoilovP, MaguireJ, DamianovA, LinCH, et al. (2011) The splicing regulator Rbfox1 (A2BP1) controls neuronal excitation in the mammalian brain. Nat Genet 43: 706–711.

69. KaynakB, von HeydebreckA, MebusS, SeelowD, HennigS, et al. (2003) Genome-wide array analysis of normal and malformed human hearts. Circulation 107: 2467–2474.

70. MartinCL, DuvallJA, IlkinY, SimonJS, ArreazaMG, et al. (2007) Cytogenetic and molecular characterization of A2BP1/FOX1 as a candidate gene for autism. Am J Med Genet B Neuropsychiatr Genet 144B: 869–876.

71. MikhailFM, LoseEJ, RobinNH, DescartesMD, RutledgeKD, et al. (2011) Clinically relevant single gene or intragenic deletions encompassing critical neurodevelopmental genes in patients with developmental delay, mental retardation, and/or autism spectrum disorders. Am J Med Genet A 155A: 2386–2396.

72. SebatJ, LakshmiB, MalhotraD, TrogeJ, Lese-MartinC, et al. (2007) Strong association of de novo copy number mutations with autism. Science 316: 445–449.

73. VoineaguI, WangX, JohnstonP, LoweJK, TianY, et al. (2011) Transcriptomic analysis of autistic brain reveals convergent molecular pathology. Nature

74. BushbyKM (1999) Making sense of the limb-girdle muscular dystrophies. Brain 122 (Pt 8)

1403–1420.

75. van der KooiAJ, de VisserM, BarthPG (1994) Limb girdle muscular dystrophy: reappraisal of a rejected entity. Clin Neurol Neurosurg 96: 209–218.

76. BeckmannJS, SpencerM (2008) Calpain 3, the “gatekeeper” of proper sarcomere assembly, turnover and maintenance. Neuromuscul Disord 18: 913–921.

77. FougerousseF, BullenP, HerasseM, LindsayS, RichardI, et al. (2000) Human-mouse differences in the embryonic expression patterns of developmental control genes and disease genes. Hum Mol Genet 9: 165–173.

78. TusherVG, TibshiraniR, ChuG (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98: 5116–5121.

79. EisenMB, SpellmanPT, BrownPO, BotsteinD (1998) Cluster analysis and display of genome-wide expression patterns. Proc Natl Acad Sci U S A 95: 14863–14868.

80. SaldanhaAJ (2004) Java Treeview–extensible visualization of microarray data. Bioinformatics 20: 3246–3248.

81. AmendolaM, VenneriMA, BiffiA, VignaE, NaldiniL (2005) Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters. Nat Biotechnol 23: 108–116.

82. NakahataS, KawamotoS (2005) Tissue-dependent isoforms of mammalian Fox-1 homologs are associated with tissue-specific splicing activities. Nucleic Acids Res 33: 2078–2089.

83. LeeJA, TangZZ, BlackDL (2009) An inducible change in Fox-1/A2BP1 splicing modulates the alternative splicing of downstream neuronal target exons. Genes Dev 23: 2284–2293.

84. KalsotraA, WangK, LiPF, CooperTA (2010) MicroRNAs coordinate an alternative splicing network during mouse postnatal heart development. Genes Dev 24: 653–658.

Štítky
Genetika Reprodukčná medicína

Článok vyšiel v časopise

PLOS Genetics


2013 Číslo 1
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Aktuální možnosti diagnostiky a léčby litiáz
nový kurz
Autori: MUDr. Tomáš Ürge, PhD.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#