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

• DOI: 10.1371/journal.pgen.1004294
• Publication Date: 2014-04-17T19:47:17Z
• Authors: Erick W. Loomis; Lionel A. Sanz; Frédéric Chédin; Paul J. Hagerman
• Keywords: FMR1; Transcription

Expansion of a trinucleotide (CGG) repeat element within the 5′ untranslated region (5′UTR) human FMR1 gene is responsible for number heritable disorders operating through distinct pathogenic mechanisms: silencing fragile X syndrome (>200 CGG) and RNA toxic gain-of-function FXTAS (∼55–200 CGG). Existing models have focused almost exclusively on post-transcriptional mechanisms, but co-transcriptional processes could also contribute to molecular dysfunction FMR1. We observed that transcription GC-rich 5′UTR favors R-loop formation, with nascent (G-rich) forming stable RNA:DNA hybrid template DNA strand, thereby displacing non-template strand. Using DNA:RNA (hybrid) immunoprecipitation (DRIP) genomic from cultured dermal fibroblasts both normal (∼30 CGG repeats) premutation (55<CGG<200 alleles, we provide evidence formation in DNA. doxycycline (DOX)-inducible episomal system which CGG-repeat frequency can be varied, further show increases higher expression levels. Finally, non-denaturing bisulfite mapping displaced single-stranded confirmed at endogenous locus indicated R-loops formed over repeats may prone structural complexities, including hairpin not commonly associated other R-loops. These observations introduce new feature directly affected by expansion likely involved cellular dysfunction.