%0 Journal Article
%T Transcription-Associated R-Loop Formation across the Human FMR1 CGG-Repeat Region
%A Erick W. Loomis
%A Fr¨¦d¨¦ric Ch¨¦din
%A Lionel A. Sanz
%A Paul J. Hagerman
%J -
%D 2014
%R 10.1371/journal.pgen.1004294
%X Expansion of a trinucleotide (CGG) repeat element within the 5¡ä untranslated region (5¡äUTR) 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¨C200 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¡äUTR 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
%K DNA methylation
%K DNA transcription
%K Fibroblasts
%K Genetic loci
%K Repeated sequences
%K DNA damage
%K DNA-RNA hybridization
%K Genetic networks
%U https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1004294