PMID- 24743386
OWN - NLM
STAT- MEDLINE
DCOM- 20141124
LR  - 20211021
IS  - 1553-7404 (Electronic)
IS  - 1553-7390 (Print)
IS  - 1553-7390 (Linking)
VI  - 10
IP  - 4
DP  - 2014 Apr
TI  - Transcription-associated R-loop formation across the human FMR1 CGG-repeat 
      region.
PG  - e1004294
LID - 10.1371/journal.pgen.1004294 [doi]
LID - e1004294
AB  - 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 ( approximately  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 ( approximately  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.
FAU - Loomis, Erick W
AU  - Loomis EW
AD  - Department of Biochemistry and Molecular Medicine, University of California, 
      Davis, School of Medicine, Davis, California, United States of America.
FAU - Sanz, Lionel A
AU  - Sanz LA
AD  - 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.
FAU - Chedin, Frederic
AU  - Chedin F
AD  - 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.
FAU - Hagerman, Paul J
AU  - Hagerman PJ
AD  - 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.
LA  - eng
GR  - R01 GM094299/GM/NIGMS NIH HHS/United States
GR  - R01 HD040661/HD/NICHD NIH HHS/United States
GR  - 5R01GM094299/GM/NIGMS NIH HHS/United States
GR  - 5R01HD040661/HD/NICHD NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20140417
PL  - United States
TA  - PLoS Genet
JT  - PLoS genetics
JID - 101239074
RN  - 0 (5' Untranslated Regions)
RN  - 0 (DNA, Single-Stranded)
RN  - 0 (FMR1 protein, human)
RN  - 139135-51-6 (Fragile X Mental Retardation Protein)
RN  - 63231-63-0 (RNA)
SB  - IM
MH  - 5' Untranslated Regions/genetics
MH  - Alleles
MH  - Cells, Cultured
MH  - DNA, Single-Stranded/genetics
MH  - Fibroblasts/metabolism
MH  - Fragile X Mental Retardation Protein/*genetics
MH  - Humans
MH  - Hybridization, Genetic/genetics
MH  - RNA/genetics
MH  - Transcription, Genetic/*genetics
MH  - Trinucleotide Repeat Expansion/*genetics
PMC - PMC3990486
COIS- I have read the journal's policy and have the following conflicts. I, Dr. 
      Hagerman, hold patents for quantification of CGG-repeat number and for 
      measurement of FMRP levels. I have submitted, with Pacific Biosciences, a patent 
      application for SMRT-sequencing methodology. I collaborate with Pacific 
      Biosciences without compensation; Pacific Biosciences and I are co-recipients of 
      an STTR grant from the NICHD.
EDAT- 2014/04/20 06:00
MHDA- 2014/12/15 06:00
PMCR- 2014/04/17
CRDT- 2014/04/19 06:00
PHST- 2013/09/10 00:00 [received]
PHST- 2014/02/21 00:00 [accepted]
PHST- 2014/04/19 06:00 [entrez]
PHST- 2014/04/20 06:00 [pubmed]
PHST- 2014/12/15 06:00 [medline]
PHST- 2014/04/17 00:00 [pmc-release]
AID - PGENETICS-D-13-02486 [pii]
AID - 10.1371/journal.pgen.1004294 [doi]
PST - epublish
SO  - PLoS Genet. 2014 Apr 17;10(4):e1004294. doi: 10.1371/journal.pgen.1004294. 
      eCollection 2014 Apr.