Project Summary Fragile X Syndrome (FXS) and Fragile X-associated/Tremor Ataxia Syndrome (FXTAS) are two FRAXopathies which are characterized by the unstable expansion of a CGG short tandem repeat (STR) located in the 5' untranslated region of the Fragile X Mental Retardation 1 (FMR1) gene. Expansion of the CGG tract from wild type length (WT, <55 CGG STR) to premutation (PM, 55-200 CGGs) results in a dramatic increase in FMR1 transcription with no noticeable elevation in levels of the protein it encodes (FMRP). Upon expansion to full mutation (FM, >200 CGGs), FMR1 is silenced via DNA methylation and, consequently, FMRP also reduces to baseline levels. Neither the removal of DNA methylation over the promoter and CGG tract nor transgene rescue cannot fully restore healthy phenotypes suggesting FMR1 and FMRP dysregulation are not the only disease drivers. The objective of my proposal is to investigate the RNA-mediated mechanisms driving disease-associated H3K9me3 deposition, trans interactions, and genome-wide STR instability in FXS. My central hypothesis is that FMR1 or FMR1-AS1 RNAs influence pathological heterochromatin deposition in a CGG length-dependent manner by toggling between sequestering key chromatin readers, writers, and erasers in inclusion bodies and forming toxic DNA:RNA structures locally and at distal loci. I have formulated my hypothesis based on our recent surprising observations that (1) Megabase-scale heterochromatin domains are acquired on autosomes and the X chromosome and spatially connect in ectopic inter-chromosomal interactions FM FXS in a manner that is dependent on the length of the CGG STR and (2) cut-back of the FM CGG to PM, or overexpression of PM-length CGG RNA, can reverse pathologic H3K9me3 deposition in FXS. Moreover, in established literature, PM-length CGG RNA forms nuclear inclusion bodies, whereas FM-length CGG can form toxic DNA-RNA R loops, but their interplay during FXS onset and progression and mechanistic connection to heterochromatin is unknown. I will test my hypothesis by employing state-of-the-art techniques like CUT&RUN, Hi-C, MapR, RADICL-seq, and ChlRP-MS in induced pluripotent stem cells differentiated to neural progenitors (iPSC-NPCs) across a range of CGG STR expansions and engineered cut-backs to shorter tracts. Upon successful completion of my experiments, I will elucidate the protein components of nuclear CGG RNA inclusion bodies, the location and sequence of RNA:DNA hybrids and R loops, heterochromatin placement, and genome folding features genome-wide as a function of CGG expansion and contraction. My work is significant because ii will elucidate the mechanisms by which FXS might progress via RNA-mediated heterochromatin in subnuclear bodies and established fundamental knowledge about the interplay between RNA-based inclusion bodies and RNA-DNA hybrids genome-wide in repeat expansion disorders. Our models of genome-wide heterochromatinization and gene silencing in FXS will also shed ligh...