Silencing of the FMR1 gene for non-coding CGG-repeat expansions in excess of 200 repeats gives rise to fragile X syndrome, the leading inherited form of intellectual disability, and to a principal single-gene form of autism. However, despite the critical importance of this epigenetic phenomenon, the mechanism(s) leading to silencing are not understood in large part due to the unavailability of tools for mapping the necessary methylation events that span both the promoter and CGG-repeat element within single alleles. We have now developed and implemented methods that will allow us to address this central epigenetic issue for the first time. The proposed research comprises three interrelated specific aims, each based on a working hypothesis and addressing a different aspect of FMR1 silencing. Specific Aim 1 (structural) is based on the hypothesis (Hypothesis 1) that methylation of the FMR1 promoter is a consequence of the initiation of methylation within the CGG repeat. This aim will be addressed using single molecule, real-time (SMRT) sequencing, which will enable us to completely define individual methylation pattern(s) across the promoter (inclusive of the CpG island) and the CGG-repeat element for a broad range of CGG-repeat-length individual alleles. Specific Aim 2 (functional) posits (Hypothesis 2) that specific epigenotypes (both mCpG and histone modifications) will be associated with differing levels of expression of FMR1 mRNA. This second aim will utilize our ability to generate multiple fibroblast sub-clones from complex mosaic individuals such that each sub-clone harbors a single epigenotype that can be matched to a specific expression level. Specific Aim 3 (mechanistic) will address the question of how methylation is triggered and will clarify the role of the CGG repeat in this process. We propose (Hypothesis 3) that co-transcriptional R-loop formation at the CGG repeat increases in frequency, length, and residence time with increasing CGG-repeat length. This in turn is proposed to lead to the formation of double-strand DNA breaks (DSBs), the repair of which triggers gene silencing through break-associated chromatin-remodeling. To test this, we will analyze R-loop formation patterns, frequency, and dynamics together with the formation of DSBs using (i) a stable, non-integrating episomal system harboring expanded CGG repeats under the control of either an inducible promoter or the native FMR1 promoter; and (ii) subclones carrying various expanded FMR1 alleles in their native chromosomal context. We expect that achieving these three aims will bring about a coherent mechanistic understanding of FMR1-gene silencing, which should in turn facilitate the development of therapeutic approaches to target, in a gene-specific fashion, elements of the silencing mechanism.