A new perspective on chromatin organizations in IPS-derived C9orf72 motor neurons
C9orf72 is the most common causative gene for amyotrophic lateral sclerosis (ALS), accounting for up to ~40% of familial and ~7% of sporadic cases. The pathological defect in the C9orf72 gene is an expansion of a hexanucleotide repeat (GGGGCC)n in the first intron (up to 1000-5000 repeats). By contrast, healthy controls typically have fewer than 30 repeats. Two hallmarks of the hexanucleotide repeat expansion (HRE) are repeat-associated sense and antisense intranuclear RNA foci and non-canonical poly dipeptide repeats. Three hypotheses to explain the pathogenicity of these repeats posit haplo-insufficiency of C9orf72 expression, deposition of intranuclear RNA foci and toxicity from the dipeptide repeats. Furthermore, in vitro studies show that HRE could form G-quadruplex structure at DNA and RNA levels. In this study, we investigated how the HRE affects higher chromatin organization locally (C9orf72 locus) and globally (genome-wide) in three cell types: fibroblasts, induced pluripotent stem cells (IPSC) and IPS-derived neurons (IPSN). These cells have been derived from two C9orf72 individuals and two age and sex-matched controls. We performed Hi-C (genome-wide chromosome conformation capture) and RNA-seq to determine how chromatin organization and transcriptome profiles in these three different cell types differ between controls and patients. Strikingly, Hi-C data showed that chromosome organization in IPSNs is dramatically different from iPSCs and fibroblasts in regards to the spatial organization and interactions of centromeric and telomeric heterochromatin. We have also detected stronger topologically associating domains and less well defined distinctions between heterochromatin and euchromatin in 2 month old IPSNs irrespective of ALS phenotype. On the other hand, we have not yet detected major changes when comparing each of the three cell types in patients to comparable cells in controls. Overall, our findings indicate that heterochromatin regulation of motor neurons is distinct from other cell types independently of disease state.