Publication Date



Open access

Embargo Period


Degree Type


Degree Name

Doctor of Philosophy (PHD)


Molecular and Cellular Pharmacology (Medicine)

Date of Defense


First Committee Member

Claes Wahlestedt

Second Committee Member

Fulvia Verde

Third Committee Member

Vladlen Slepak

Fourth Committee Member

Derek Dykxhoorn

Fifth Committee Member

Alejandro Caicedo


Hexanucleotide repeat expansion in C9ORF72 gene has recently been shown to cause familial amyotrophic lateral sclerosis, a neurodegenerative disease caused by global death of motor neurons. The expansion leads to partial heterochromatinization of the locus, yet mutant RNAs and dipeptide repeat proteins are still produced in sufficient quantities to confer neurotoxicity. So far several research groups have identified DNA hypermethylation at C9ORF72 promoter CpG sites in a fraction of patients, but the developmental timing and the reason of its occurrence only in a subset of individuals remains unknown. In order to model the acquisition of C9ORF72 hypermethylation, we generated induced pluripotent stem cells from an ALS patient with C9ORF72 promoter hypermethylation. Our data show that methylation levels are reduced by reprogramming and then re-acquired upon neuronal specification, while hydroxymethylation levels increase following reprogramming and are highest in iPSCs and motor neurons. We confirmed the presence of hydroxymethylation within the C9ORF72 promoter in post-mortem brain tissues of hypermethylated patients. Using iPSC neurons, we found that preventing R-loop formation did not impede heterochromatinization of the expanded locus. Moreover, we show that in C9-BAC mouse model of ALS, partial heterochromatinization of the C9ORF72 occurs during the first weeks of the lifespan, indicating that epigenetic repression is developmentally regulated. Taken together, these observations provide further insight into mechanism and developmental time-course of epigenetic perturbations conferred by the C9ORF72 repeat expansion. The Fragile X Syndrome (FXS) results from a repeat expansion mutation near the FMR1 gene promoter and is the most common form of heritable intellectual disability and autism. Mutations larger than 200 CGG repeats trigger FMR1 heterochromatinization and loss of gene expression, which is primarily responsible for the pathological features of FXS. While the role of 5-methylcytosine (5mC) in FMR1 gene silencing has been studied extensively, the role of 5-hydroxymethylation (5hmC), a newly discovered epigenetic mark produced through active DNA demethylation, has not been previously investigated in FXS neurons. Here, we used two complementary epigenetic assays, hydroxymethylation sensitive restriction digest and TET-assisted bisulfite pyrosequencing, to quantify FMR1 5mC and 5hmC levels. We observed increased levels of 5hmC at the FMR1 promoter in FXS patient brains with full-mutations relative to pre-mutation carriers and unaffected controls. In addition, we found that 5hmC enrichment at the FMR1 locus in FXS cells is specific to neurons by utilizing a nuclei sorting technique to separate neuronal and glial DNA fractions from post-mortem brain tissues. Future studies could investigate the potential to leverage this epigenetic pathway to restore FMR1 expression and discern whether levels of 5hmC correlate with phenotypic severity.


C9ORF72-ALS; Fragile X Syndrome; iPSC; DNA hydroxymethylation; epigenetics