Spatially coordinated heterochromatinization of distal short tandem repeats in fragile X syndrome

Abstract

Short tandem repeat (STR) instability is causally linked to pathologic transcriptional silencing in a subset of repeat expansion disorders. In fragile X syndrome (FXS), instability of a single CGG STR tract is thought to repress FMR1 via local DNA methylation. Here, we report the acquisition of more than ten Megabase-sized H3K9me3 domains in FXS, including a 5-8 Megabase block around FMR1. Distal H3K9me3 domains encompass synaptic genes with STR instability, and spatially co-localize in trans concurrently with FMR1 CGG expansion and the dissolution of TADs. CRISPR engineering of mutation-length FMR1 CGG to normal-length preserves heterochromatin, whereas cut-out to pre-mutation-length attenuates a subset of H3K9me3 domains. Overexpression of a pre-mutation-length CGG de-represses both FMR1 and distal heterochromatinized genes, indicating that long-range H3K9me3-mediated silencing is exquisitely sensitive to STR length. Together, our data uncover a genome-wide surveillance mechanism by which STR tracts spatially communicate over vast distances to heterochromatinize the pathologically unstable genome in FXS.One-Sentence SummaryHeterochromatinization of distal synaptic genes with repeat instability in fragile X is reversible by overexpression of a pre-mutation length CGG tract.

MASTR-seq: Multiplexed Analysis of Short Tandem Repeats with sequencing

“…In our recently published work focused on fragile X syndrome (FXS) 48,49 , we applied our method to accurately measure CGG STR length and DNA methylation for the 5'UTR and promoter of the FMR1 gene in the same single-allele across several pooled normal-length, premutation-length, and mutation-length patient-derived iPSC lines. By enriching the proportion of long-reads spanning the target locus by >10-fold compared to the nanopore Cas9-targeted sequencing (nCAT) method 33 , we achieved >300x on-target read coverage at the FMR1 CGG STR in a pool of 8-12 independent samples in a single MinION flow cell (BOX6).…”

Narrative Review: Update on the Molecular Diagnosis of Fragile X Syndrome

“…They also discovered a large group of genes distal from FMR1 that are repressed in FXS in concordance with H3K9me3 deposition. The distal genes have an important role in synaptic plasticity, testis development, and the reproductive system, which coincide with the phenotype of FXS [95] Grosso et al tried a different approach and combined short-read sequencing (Illumina) with long-read sequencing (Nanopore). They tried to combine the advantages of shortread and long-read sequencing.…”

3D Genome Plasticity in Normal and Diseased Neurodevelopment

“…Ultimately, abnormal regulation across multiple epigenomic layers then very likely contributes to abnormal expression and silencing of the transcript for the fragile X mental retardation protein (FMRP), a widely expressed RNA-binding protein essential for proper synaptic plasticity and architecture [ 49 ]. Even more remarkably, these localized alterations in 3D chromatin structure at the Fragile X locus result in excessive spreading of heterochromatin upstream of the disease-associated STR, with the abnormal heterochromatic spread engulfing two X-linked neuronal cell adhesion genes, SLTIRK2 and SLTIKR4 [ 50 ], in addition to various autosomal loci interconnected in trans with the Fragile X locus, altogether affecting more than 10 Mb of genome sequence [ 50 ]. Therefore, similarly to the microdeletion syndromes, chromosomal conformation changes at neurologically relevant short tandem repeat expansions are not limited to the affected site-specific locus, but also affect additional loci that are in spatial 3D proximity to the specific repeat expansion site.…”