# Elucidating the spatially coordinated mechanisms of transcriptional silencing in fragile X syndrome

> **NIH NIH F30** · UNIVERSITY OF PENNSYLVANIA · 2024 · $53,974

## Abstract

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have formulated my hypothesis based on my unpublished imaging data demonstrating that (1) ectopic trans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...

## Key facts

- **NIH application ID:** 10824676
- **Project number:** 1F30HD114405-01
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Kenneth Pham
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $53,974
- **Award type:** 1
- **Project period:** 2024-03-01 → 2028-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10824676

## Citation

> US National Institutes of Health, RePORTER application 10824676, Elucidating the spatially coordinated mechanisms of transcriptional silencing in fragile X syndrome (1F30HD114405-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10824676. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*