# Connecting 3D genome misfolding to transcriptional silencing in fragile X syndrome > **NIH NIH R37** · UNIVERSITY OF PENNSYLVANIA · 2024 · $541,806 ## Abstract Fragile X syndrome (FXS) is an inherited neurodevelopmental disorder associated with social anxiety, hypersensitivity to stimuli, seizures, and learning disabilities. It is classically viewed as a monogenic disease in which mutation-length expansion of a CGG short tandem repeat (STR) tract above a critical mutation-length threshold of 200 triplets leads to local DNA methylation and repression of FMR1. Genome-wide gene expression changes are thought to occur downstream of the loss of the Fragile X Messenger Ribonucleoprotein (FMRP) encoded by FMR1. In our lab’s new data acquired during the previous funding period, we used nanopore long- read sequencing, Hi-C, CUT&RUN, CRISPR STR engineering, and single-cell Oligopaint imaging to find Megabase (Mb)-sized H3K9me3 domains on autosomes and the X-chromosome in FXS patient-derived cell lines and brain tissue. Domains co-localize with severe misfolding of topologically associating domains (TADs) and loops and connect via trans interactions in subnuclear hubs. Because H3K9me3 domains encompass STRs susceptible to instability and replication stress-induced double strand breaks, we termed them BREACHes: Beacons of Repeat Expansion Anchored by Contacting Heterochromatin. BREACHes encompass and silence genes encoding synaptic plasticity, epithelial integrity, reproductive development, and neural cell adhesion, which are clinical hallmarks in FXS. Thus, by way of Mb-scale heterochromatin domains and trans interactions, we find multiple plausible candidate genes of possible relevance for understanding onset, progression or therapeutic treatment of FXS. The objective of this proposal is to elucidate the RNA-mediated and higher-order folding mechanisms governing heterochromatin domains and the trans interactions among them in FXS. Our central hypothesis is that mutation-length CGG STR-containing RNA forms RNA:DNA hybrid structures to establish H3K9me3 domains, whereas the attenuation of cohesin-mediated loop extrusion is necessary to maintain H3K9me3 and trans interactions. We will test our hypothesis with two Specific Aims. First, we will investigate the role for CGG STR-containing RNA in BREACH establishment. To model the removal of H3K9me3 signal and the subsequent re-establishment of the domains, we will reprogram normal-length, FXS mutation- length, and premutation-length cutback induced pluripotent stem cells (iPSCs) from primed to naïve pluripotency and release back to primed. In a timecourse of BREACH re-establishment, we will assay R loops, RNA-chromatin interactions, histone methyl transferases, and 3D genome folding. Second, we will elucidate the effect of cohesin- mediated loop extrusion on BREACH maintenance in single cells. We will deplete and replete the cohesin unloading factor WAPL in FXS iPSCs. In a timecourse of recovery and loss of cohesin-mediated loop extrusion, we will assay genome folding, DNA methylation, and H3K9me3 with cutting edge single-cell genomics and imaging technologies. Togethe... ## Key facts - **NIH application ID:** 10983660 - **Project number:** 2R37MH120269-06 - **Recipient organization:** UNIVERSITY OF PENNSYLVANIA - **Principal Investigator:** Jennifer Elizabeth Phillips-Cremins - **Activity code:** R37 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2024 - **Award amount:** $541,806 - **Award type:** 2 - **Project period:** 2019-08-15 → 2029-01-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10983660 ## Citation > US National Institutes of Health, RePORTER application 10983660, Connecting 3D genome misfolding to transcriptional silencing in fragile X syndrome (2R37MH120269-06). Retrieved via AI Analytics 2026-06-02 from https://api.ai-analytics.org/grant/nih/10983660. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*