Transcription-associated R-loop formation across the human FMR1 CGG-repeat region

PLoS Genet. 2014 Apr 17;10(4):e1004294. doi: 10.1371/journal.pgen.1004294. eCollection 2014 Apr.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 5' Untranslated Regions / genetics
  • Alleles
  • Cells, Cultured
  • DNA, Single-Stranded / genetics
  • Fibroblasts / metabolism
  • Fragile X Mental Retardation Protein / genetics*
  • Humans
  • Hybridization, Genetic / genetics
  • RNA / genetics
  • Transcription, Genetic / genetics*
  • Trinucleotide Repeat Expansion / genetics*

Substances

  • 5' Untranslated Regions
  • DNA, Single-Stranded
  • FMR1 protein, human
  • Fragile X Mental Retardation Protein
  • RNA