Elucidating the interplay between cohesin-mediated loop extrusion and heterochromatin in single cells. - Project Summary Fragile X Syndrome (FXS) is characterized by the unstable expansion of a CGG short tandem repeat (STR) located in the 5’ untranslated region of the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene. Expansion of the CGG tract from normal-length (WT, <55 CGG) to mutation-length (ML, >200 CGG) results in silencing of FMR1 transcription and severe loss of the protein it encodes. Textbook models of FXS have long attributed FMR1 silencing to local DNA methylation over the promoter and CGG tract, however removal of methylation is insufficient to reproducibly de-repress the gene in many experiments. Recently, my lab discovered a Megabase- scale domain of H3K9me3 on the X-chromosome and multiple autosomes in FXS induced pluripotent stem cell (iPSC) lines with a mutation-length CGG tract. The H3K9me3 domains encompass repressed synaptic genes, including FMR1, and severe misfolding of the 3D genome. The overall objective of my proposal is to investigate the interplay between cohesin-mediated loop extrusion, H3K9me3, and gene expression in FXS at the resolution of single-cells. Progress toward testing this goal has been limited by the paucity of single-cell approaches for mapping cohesin occupancy and chromatin folding at kilobase-resolution. My central hypothesis is that the FMR1 locus can undergo cohesin-mediated loop extrusion in WT-iPSCs. By contrast, I posit that ML-FXS iPSCs with H3K9me3 domains will exhibit depleted cohesin binding that is functionally linked to severely misfolded topologically associating domains (TADs), subTADs, and loops and silenced FMR1 expression. I generated my hypotheses based on (1) published work demonstrating that depletion of the cohesin unloading factor WAPL causes hyper-extrusion, stabilization of cohesin binding, and loss of H3K9me3 signal, and (2) my own preliminary data showing that loops are abolished at H3K9me3 domains in ML-FXS iPSCs and rescued when H3K9me3 signal is reversed. I will test my hypothesis by employing both single-cell imaging and genomics techniques to map genome folding, immunofluorescence staining of cohesin and H3K9me3, and single-cell TIPseq to assay cohesin binding and H3K9me3 in WT and ML-FXS iPSCs. I will also utilize WAPL degron iPSC lines established in my lab in ML genetic backgrounds to assay changes in genome folding due to loss and gain of cohesin extrusion at the FMR1 locus. Upon successful completion of my experiments, I will have elucidated mechanisms underlying the interplay between chromatin folding and H3K9me3 maintenance in FXS. My work is significant because knowledge generated in this proposal will have impact on our understanding of how heterochromatin disrupts gene expression in a wide range of neurodevelopmental and neurodegenerative disorders. In addition, I will create freely available algorithms to quantify cohesin-mediated loop extrusion from chromatin tracing data.
