SRP409538 PRJNA904800 GSE218679 Spatially coordinated heterochromatinization of long synaptic genes in fragile X syndrome [Nanopore] Short tandem repeat (STR) instability causes transcriptional silencing in several repeat expansion disorders. In fragile X syndrome (FXS), mutation-length expansion of a CGG STR represses FMR1 via local DNA methylation. Here, we find Megabase-scale H3K9me3 domains on autosomes and encompassing FMR1 on the X-chromosome in induced pluripotent stem cell (iPSC)-derived neural progenitors, B-cells, and fibroblasts with mutation-length CGG expansion. H3K9me3 domains connect via inter-chromosomal interactions and demarcate severe misfolding of TADs and loops. They harbor long synaptic genes replicating at the end of S phase, replication stress-induced double strand breaks, and STRs prone to stepwise somatic instability. CRISPR engineering of the full-mutation CGG to premutation-length reverses H3K9me3 domains on the X-chromosome and multiple autosomes, refolds TADs, and restores expression. H3K9me3 domains also arise in a subset of normal-length iPSCs with increased STR instability burden. Our results reveal Mb-scale heterochromatinization and trans interactions among chromosomes susceptible to repeat genetic instability. Overall design: Hi-C, H3K9me3 ChIP-seq, CTCF ChIP-seq, RNA-seq, H3K9me3 CUT&RUN, and short and long read genome sequencing were used in multiple cell types to interrogate epigenetic changes in cell lines with normal, premutation, and disease level repeat expansions in FMR1. GSE218679 pubmed 38134876 parent_bioproject PRJNA904794