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

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

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

Erick W Loomis et al. PLoS Genet..

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.

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Conflict of interest statement

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.

Figures

Figure 1
Figure 1. Sequence analysis of theFMR1 promoter reveals signatures of R-loop formation.
GC skew (red, left y-axis), CpG observed/expected ratio (CpG O/E; navy, right y-axis), and GC% (gray, right y-axis) calculated over a sliding 100 nt window from −500 to +1200 nt around the downstream-most known transcription start site (vertical dotted line). Gray-shaded box highlights CGI defined by CpG O/E>0.6 (navy dotted lines) and GC%>50% for at least 200 nt. Schematic at the top shows theFMR1 5′UTR with multiple transcription start sites (black arrows), G-clusters (red ticks), and CGG repeats (striped box), all overlapping the CGI (gray bar) for scale to the graph below.
Figure 2
Figure 2. R-loop pull-down in human dermal fibroblasts confirms R-loop formation in the genome.
(A) Fold enrichment forFMR1 in dermal fibroblast cells cultured from seven individuals using a monoclonal antibody specific to hybrids. Enrichment is relative to input and a non-R-loop-forming genomic reference locus. (B) Treatment with recombinant RNases H1 and H2 (RNase H) eliminates enrichment seen forFMR1 (solid lines) andMYADM (broken lines).
Figure 3
Figure 3. Effect of transcription and repeat length onFMR1 R-loop formation.
(A) Schematic of DOX-ON constructs with short or expandedFMR1 CGG repeats or non-FMR1 sequence, each with GFP reporter tags. Black arrowheads mark sites of restriction enzyme cleavage prior to DRIP, with EcoRI cutting at the start of theFMR1 5′UTR and XbaI cutting at the end ofEGFP. (B) mRNA expression relative to non-induced cells for each construct. Error bars: SEM from 2 biological replicates. (C) DRIP fold enrichment of GFP fragment relative to the episome backbone. Error bars: SEM from 3 biological replicates. (D) DRIP percentage of input normalized to peak recovery (6 hours DOX ON) of GFP fragment at 0, 1, 2, and 24 hours post DOX washout, and No-DOX treatment. Error bars: SEM from 3 biological replicates.
Figure 4
Figure 4. Non-denaturing bisulfite footprinting of the displaced DNA strand of theFMR1 R-loop.
Each row represents an individual sequence clone, grouped together for each allele size, from cultured human dermal fibroblasts. Each column is a cytosine position, with filled boxes representing converted, single-stranded DNA and open boxes representing unconverted, double-stranded DNA. Empty boxes represent sequence gaps from bacterial deletion or loss of clean sequencing signal. Schematic diagram at the top represents theFMR1 5′UTR with marked TSSs (black arrows), translation start (ATG), CGG repeats (striped box with orange border), PCR primers (blue arrows), and G-clusters (red ticks; red dotted lines).
Figure 5
Figure 5. Model of proposed CGG-repeat effects on theFMR1 R-loop.
R-loops that span theFMR1 CGG-repeat region (yellow) during transcription could adopt a hairpin structure within the displaced CGG-repeat strand, thus protecting the CGG-repeat region from bisulfite conversion while leaving both 5′ and 3′ flanking regions exposed; the CGG-repeat is known to form such structures readilyin vitro. An alternative structure, although less energetically feasible, would involve maintenance of R-loops flanking the CGG-repeat element, which has collapsed into a dsDNA structure again. Loss of the upstream R-loop region would explain the absence of bisulfite conversion in ∼25–50% of molecules (Figure 4). Red, nascent RNA transcript; 90° arrow, start of transcription; blue sphere, Pol II.
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