title: Transcription-associated R-loop formation across the human FMR1 CGG-repeat region.
abstract: Expansion of a trinucleotide (CGG) repeat element within the 5' untranslated region (5'UTR) of the human FMR1 gene is responsible for a number of heritable disorders operating through distinct pathogenic mechanisms: gene silencing for fragile X syndrome (>200 CGG) and RNA toxic gain-of-function for FXTAS (∼ 55-200 CGG). Existing models have focused almost exclusively on post-transcriptional mechanisms, but co-transcriptional processes could also contribute to the molecular dysfunction of FMR1. We have observed that transcription through the GC-rich FMR1 5'UTR region favors R-loop formation, with the nascent (G-rich) RNA forming a stable RNA:DNA hybrid with the template DNA strand, thereby displacing the non-template DNA strand. Using DNA:RNA (hybrid) immunoprecipitation (DRIP) of genomic DNA from cultured human dermal fibroblasts with both normal (∼ 30 CGG repeats) and premutation (55<CGG<200 repeats) alleles, we provide evidence for FMR1 R-loop formation in human genomic DNA. Using a doxycycline (DOX)-inducible episomal system in which both the CGG-repeat and transcription frequency can be varied, we further show that R-loop formation increases with higher expression levels. Finally, non-denaturing bisulfite mapping of the displaced single-stranded DNA confirmed R-loop formation at the endogenous FMR1 locus and further indicated that R-loops formed over CGG repeats may be prone to structural complexities, including hairpin formation, not commonly associated with other R-loops. These observations introduce a new molecular feature of the FMR1 gene that is directly affected by CGG-repeat expansion and is likely to be involved in the associated cellular dysfunction.
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, California, United States of America.
authors.forename: Erick W
authors.initials: EW
authors.lastname: Loomis
authors.affiliation: Department of Molecular and Cellular Biology, University of California, Davis, Davis, California, United States of America; The Genome Center, University of California, Davis, Davis, California, United States of America.
authors.forename: Lionel A
authors.initials: LA
authors.lastname: Sanz
authors.affiliation: Department of Molecular and Cellular Biology, University of California, Davis, Davis, California, United States of America; The Genome Center, University of California, Davis, Davis, California, United States of America.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Chédin
authors.affiliation: Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Davis, California, United States of America; MIND Institute, University of California, Davis, Health System, Sacramento, California, United States of America.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hagerman
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D000483:Alleles; D002478:Cells, Cultured; D004277:DNA, Single-Stranded / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics*; D006801:Humans; D006824:Hybridization, Genetic / Q000235:genetics; D012313:RNA / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D019680:Trinucleotide Repeat Expansion / Q000235:genetics*
chemical_list: D020121:5' Untranslated Regions; D004277:DNA, Single-Stranded; D051860:Fragile X Messenger Ribonucleoprotein 1; D012313:RNA; C497683:FMR1 protein, human
references.citation: Bioinformatics. 2007 Nov 1;23(21):2947-8
references.pmid: 17846036
references.citation: Acta Neuropathol. 2013 Jul;126(1):1-19
references.pmid: 23793382
references.citation: Nucleic Acids Res. 2011 Aug;39(14):6172-85
references.pmid: 21478165
references.citation: Mol Cell. 2011 Jun 24;42(6):794-805
references.pmid: 21700224
references.citation: Cell. 1990 Jul 27;62(2):331-8
references.pmid: 1695550
references.citation: Nucleic Acids Res. 2008 Feb;36(3):1050-6
references.pmid: 18160412
references.citation: PLoS Genet. 2007 Jul;3(7):e110
references.pmid: 17616978
references.citation: RNA Biol. 2007 Sep;4(2):93-100
references.pmid: 17962727
references.citation: Hum Mol Genet. 2010 Apr 15;19(R1):R83-9
references.pmid: 20430935
references.citation: Proc Natl Acad Sci U S A. 2006 Mar 28;103(13):5030-5
references.pmid: 16547142
references.citation: Mol Cell. 2011 Dec 23;44(6):966-77
references.pmid: 22195969
references.citation: Cell. 1995 May 19;81(4):533-40
references.pmid: 7758107
references.citation: Mol Cell. 2012 Mar 30;45(6):814-25
references.pmid: 22387027
references.citation: Proc Natl Acad Sci U S A. 2013 Aug 20;110(34):13938-43
references.pmid: 23918391
references.citation: Am J Hum Genet. 2000 Jan;66(1):6-15
references.pmid: 10631132
references.citation: Hum Mol Genet. 2010 Jan 15;19(2):299-312
references.pmid: 19864489
references.citation: PLoS One. 2011;6(8):e23648
references.pmid: 21909353
references.citation: J Neurodev Disord. 2011 Sep;3(3):211-24
references.pmid: 21617890
references.citation: Arch Neurol. 2008 Jan;65(1):19-25
references.pmid: 18195136
references.citation: Genes Dev. 2012 Jan 15;26(2):163-75
references.pmid: 22279048
references.citation: J Biol Chem. 2011 Mar 25;286(12):10530-9
references.pmid: 21177854
references.citation: EMBO J. 2010 Apr 7;29(7):1248-61
references.pmid: 20186122
references.citation: J Mol Diagn. 2005 Nov;7(5):605-12
references.pmid: 16258159
references.citation: RNA Biol. 2010 Sep-Oct;7(5):551-8
references.pmid: 20729633
references.citation: Biochem J. 2010 Aug 1;429(3):545-52
references.pmid: 20513237
references.citation: Mol Cell. 2009 Jul 31;35(2):228-39
references.pmid: 19647519
references.citation: Nat Genet. 1994 Sep;8(1):88-94
references.pmid: 7987398
references.citation: Mol Cell. 2003 Sep;12(3):711-21
references.pmid: 14527416
references.citation: Nucleic Acids Res. 2009 Jul;37(13):4385-92
references.pmid: 19465392
references.citation: Nat Protoc. 2008;3(6):1101-8
references.pmid: 18546601
references.citation: J Mol Biol. 1994 Oct 21;243(2):143-51
references.pmid: 7932745
references.citation: Hum Mol Genet. 2011 Jun 1;20(11):2161-70
references.pmid: 21389081
references.citation: Science. 1991 May 24;252(5009):1097-102
references.pmid: 2031184
references.citation: Genome Res. 2013 Oct;23(10):1590-600
references.pmid: 23868195
references.citation: Mol Cell Biol. 2008 Jan;28(1):50-60
references.pmid: 17954560
references.citation: Mol Cell. 2012 Apr 27;46(2):115-24
references.pmid: 22541554
references.citation: J Child Neurol. 2014 Mar;29(3):326-30
references.pmid: 23266944
references.citation: Nucleic Acids Res. 2011 Mar;39(5):1749-62
references.pmid: 21051337
references.citation: Cell Cycle. 2010 Jan 15;9(2):274-8
references.pmid: 20023421
references.citation: Cell Rep. 2013 Mar 28;3(3):869-80
references.pmid: 23478018
references.citation: FEBS J. 2009 Mar;276(6):1494-505
references.pmid: 19228196
references.citation: EMBO Rep. 2009 Aug;10(8):887-93
references.pmid: 19557000
references.citation: Nat Immunol. 2003 May;4(5):442-51
references.pmid: 12679812
references.citation: Proc Natl Acad Sci U S A. 1995 May 23;92(11):5199-203
references.pmid: 7761473
references.citation: Hum Mol Genet. 2004 Mar 1;13(5):543-9
references.pmid: 14722156
references.citation: Semin Reprod Med. 2011 Jul;29(4):299-307
references.pmid: 21969264
references.citation: Mol Cell. 2003 Mar;11(3):807-15
references.pmid: 12667461
references.citation: J Mol Biol. 2011 Jun 24;409(5):758-72
references.pmid: 21549127
references.citation: J Mol Biol. 1987 Jul 20;196(2):261-82
references.pmid: 3656447
references.citation: Am J Hum Genet. 2009 Nov;85(5):606-16
references.pmid: 19853235
references.citation: Nature. 2001 Feb 15;409(6822):860-921
references.pmid: 11237011
references.citation: Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16899-903
references.pmid: 12477932
references.citation: Neuron. 2013 May 8;78(3):440-55
references.pmid: 23602499
references.citation: Mol Cell Biol. 2009 Jun;29(11):3124-33
references.pmid: 19307304
references.citation: J Immunol Methods. 1986 May 1;89(1):123-30
references.pmid: 2422282
references.citation: Cell. 1991 Dec 20;67(6):1047-58
references.pmid: 1760838
references.citation: BMC Genomics. 2010 Feb 10;11 Suppl 1:S1
references.pmid: 20158866
references.citation: Mol Cell. 2011 Jan 7;41(1):21-32
references.pmid: 21211720
references.citation: Nucleic Acids Res. 2009 Oct;37(19):6371-7
references.pmid: 19710035
references.citation: Nat Struct Mol Biol. 2013 Apr;20(4):412-8
references.pmid: 23552296
references.citation: Adv Pediatr. 2009;56:165-86
references.pmid: 19968948