Molecular Consequences of Expanded CGG Repeats in the Human FMR1 Gene: R-loops, Methylation, and DNA Sequencing

Expansion of a CGG trinucleotide repeat sequence in the 5' untranslated region (5'UTR) of the human fragile X mental retardation 1 (FMR1) gene results in multiple human disorders with distinct molecular pathologies. Alleles larger than ~200 CGG repeats give rise to fragile X syndrome (FXS), the most common heritable form of intellectual disability, via gene silencing and hypermethylation of the FMR1 CpG island (CGI) promoter. Alleles in the ∼55--200 CGG-repeat ("premutation") range confer increased risk of matrilineal transmission of "full-mutation" offspring with FXS. However, premutation alleles are also responsible for a family of disorders that is distinct from FXS; namely, fragile X-associated tremor/ataxia syndrome (FXTAS), primary ovarian insufficiency (FXPOI), and neurodevelopmental problems. Disorders of premutation and full mutation alleles arise through completely separate mechanisms, whereby increased expression of toxic expanded-repeat mRNA causes FXTAS/FXPOI, and loss of mRNA/protein expression causes FXS.;In this dissertation, I have focused on events involving transcription of the FMR1 gene and the molecular consequences of the CGG repeat expansion.;RNA:DNA hybrid R-loop structures form in the human genome when the nascent transcript rehybridizes with the template DNA strand, displacing the non-template strand. Genomic R-loop formation is involved with transcription termination and protection of CGI promoters from DNA methylation. R-loops are also required for initiation of class-switch recombination and inappropriate R-loop formation, resulting from excess DNA supercoiling or reduced RNA processing, activates the DNA damage response. I present definitive evidence of R-loop formation at the FMR1 locus in human genomic DNA and in a model system for transcribed CGG toxicity. Repeat expansion results in increased R-loop formation at FMR1 and appears to shift the FMR1 R-loop away from its normal function into the molecular pathology of repeat instability, and the RNA-mediated cellular toxicity of FXTAS.;To further characterize the FMR1 CGG repeats, I applied newly developed single molecule real-time (SMRT) sequencing to this region. In collaboration with Pacific Biosciences, I was able to sequence large CGG-repeat alleles well into the full mutation range (∼750 CGG) and investigate the nature of CGG-repeat DNA samples generated by PCR and molecular cloning. This study also revealed a unique kinetic signature for DNA polymerase inside CGG repeat DNA, which has implications for our understanding of trinucleotide repeat replication and for future studies utilizing polymerase kinetics to detect epigenetic modifications. I then applied this sequencing methodology to map DNA methylation inside expanded CGG repeats with sodium bisulfite. This represents the highest resolution examination of methylation inside the CGG repeats to date. These data show intermediate methylation patterns that are potentially indicative of the mechanism by which this region is hypermethylated in FXS. Overall, this dissertation is a significant step forward in our understanding of the human FMR1 gene and the molecular events related to transcription of expanded CGG repeats that lead alternately to cellular toxicity, repeat instability, and/or gene silencing.