Transcription-Associated R-Loop Formation across the Human FMR1 CGG-Repeat Region

Erick W. Loomis, Lionel A. Sanz, Frederic Chedin, Paul J Hagerman

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Article numbere1004294
JournalPLoS Genetics
Volume10
Issue number4
DOIs
StatePublished - 2014

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transcription (genetics)
DNA
RNA
5' Untranslated Regions
5' untranslated regions
gene
genomics
Trinucleotide Repeats
Fragile X Syndrome
bisulfites
single-stranded DNA
doxycycline
Doxycycline
Poisons
Single-Stranded DNA
Gene Silencing
gene silencing
Immunoprecipitation
Genes
fibroblasts

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Ecology, Evolution, Behavior and Systematics
  • Cancer Research
  • Genetics(clinical)
  • Medicine(all)

Cite this

Transcription-Associated R-Loop Formation across the Human FMR1 CGG-Repeat Region. / Loomis, Erick W.; Sanz, Lionel A.; Chedin, Frederic; Hagerman, Paul J.

In: PLoS Genetics, Vol. 10, No. 4, e1004294, 2014.

Research output: Contribution to journalArticle

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