Connecting 3D genome misfolding to transcriptional silencing in fragile X syndrome

Title: Connecting 3D genome misfolding to transcriptional silencing in fragile X syndrome Project Summary/Abstract More than 30 inherited neurological disorders, including fragile X syndrome (FXS), Huntington's disease, amyotrophic lateral sclerosis, and Friedreich's ataxia, are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). In FXS, STR expansion above a critical length threshold causes pathogenic silencing of the STR-containing gene, resulting in severe neuropsychiatric symptoms. An increased understanding of the molecular mechanisms governing how STR expansion contributes to transcriptional silencing would facilitate efforts to develop treatments for repeat expansion disorders driven by pathologic gene expression changes. The objective of this proposal is to understand the link among higher-order chromatin architecture, repressive chromatin modifications, architectural protein occupancy, and gene expression silencing in FXS. Recently, we discovered that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic instability or topological disruption upon STR expansion. Consistent with this idea, we found that FXS patients exhibit severe boundary disruption and loss of CTCF occupancy in a manner that correlates with the degree of FMR1 silencing. Due to the boundary disruption, the FMR1 gene undergoes a topological switch from the downstream domain containing numerous putative enhancers to the upstream domain devoid of regulatory elements. Based on these findings, we hypothesized that 3D genome miswiring is causally linked to pathologic silencing of FMR1 via the gene's topological switch from an active to silenced regulatory landscape. We will test our hypothesis with three Specific Aims. First, we will dissect the cause-and-effect link between 3D genome misfolding and FMR1 silencing. We will assay architecture and FMR1 expression after mutating CTCF and YY1 binding sites at the FMR1 boundary with CRISPR-Cas9 and ectopically silencing FMR1 with the dCas9-KRAB repressor in healthy cells. Second, we will create genome-wide maps of CTCF/YY1 binding, H3K9me3/H3K27me3 repressive domains, and DNA methylation in FXS samples with a range of CGG daSTR lengths. By computationally integrating this data, we will elucidate how STR tract length affects distal epigenetic modifications to disrupt chromatin architecture and FMR1 expression. Third, we will re-engineer genome topology in FXS patient cells via synthetic architectural proteins and active de-methylation of specific architectural motifs. We will determine the degree to which 3D genome engineering alone or in combination with linear Epigenome engineering will reprogram repressive chromatin marks and de-repress FMR1. Our work is significant because it will shed new light into how 3-D epigenetic mechanisms go awry and can be repaired during the acquisition and progression of neurodevelopmental and neurodegenerative disease states.