Project Summary Fragile X Syndrome (FXS) is caused by expansion of the CGG short tandem repeat (STR) located in the 5’ UTR of the FMR1 gene. Upon expansion to mutation-length, local DNA methylation at the FMR1 promoter leads to silenced transcription which is thought to drive the pathophysiology of FXS. However, Fmr1 knock-out mice do not reproducibly recapitulate the range of FXS clinical presentations, suggesting that FMR1 dysregulation alone cannot explain the pathophysiology of FXS. Recently, our lab uncovered Megabase-scale domains of the histone modification H3K9me3 at the FMR1 locus on chromosome X and multiple autosomes. The H3K9me3 domains encompass silenced genes encoding synaptic plasticity and epithelial integrity, which correlate with symptoms experiences by FXS patients, raising the possibility that these heterochromatin domains contribute to the pathophysiology of FXS. The objective of my proposal is to investigate the mechanisms by which the mutation- length CGG STR coordinates Megabase-scale heterochromatin domains and their inter-chromosomal contacts. My central hypothesis is that expansion of CGG STR to mutation-length is necessary and sufficient for the heterochromatinization of the FMR1 locus and a subset of autosomal domains. Upon heterochromatinization, the domains form pathologic inter-chromosomal contacts with each other in a H3K9me3-dependent manner. I have formulated my hypothesis based on my unpublished imaging data demonstrating that (1) ectopic trans interactions form between H3K9me3 domains in induced pluripotent stem cells (iPSCs) with the mutation-length CGG STR tract, (2) in single cells, FXS domains that form inter-chromosomal contacts are more enriched in H3K9me3 than those that do not, and (3) cut-back of the CGG STR can reverse H3K9me3 signal at a subset of domains. I will test my hypothesis by leveraging a newly developed protocol for STR synthesis with CRISPR/Cas9 engineering to generate iPSC clones with the same genetic background but varying STR length and sequence at the 5’ UTR of FMR1. I will measure the effect of the mutation-length CGG STR, as well as the overexpression of H3K9me3 writer and eraser enzymes, on H3K9me3, trans interactions, and transcriptional silencing using state-of-the art genomics and multimodal imaging techniques including CUT&Run, Hi-C, sequential Oligopaint FISH, and single-molecule RNA FISH. Successful completion of these experiments will demonstrate the contribution of both STR sequence and length to the multi-chromosomal, Megabase-scale heterochromatinization of the FXS genome and defined the requirement for H3K9me3 for trans interactions. My work is significant because it will expand classic models of how the mutation-length CGG STR causes FXS to include Megabase-scale heterochromatinization and ectopic inter-chromosomal contacts. Studying these mechanisms will have a broad impact on our understanding of the role for heterochromatin and miswiring of the 3D genome in a wide ra...