The FMR1 promoter is selectively hydroxymethylated in primary neurons of fragile X syndrome patients

Abstract

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. Full mutations larger than 200 CGG repeats trigger FMR1 heterochromatinization and loss of gene expression, which is primarily responsible for the pathological features of FXS . In contrast, smaller pre-mutations of 55–200 CGG are associated with FMR1 overexpression and Fragile X-associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative condition. 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, 5hmC sensitive restriction digest and ten-eleven translocation-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. This FMR1 5hmC enrichment was not present in cellular models of FXS including fibroblasts, lymphocytes and reprogrammed neurons, indicating they do not fully recapitulate this epigenetic feature of disease. 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.

Intragenic DNA methylation in buccal epithelial cells and intellectual functioning in a paediatric cohort of males with fragile X

“…The observed increase in BEC FREE2 DNA methylation extends previous studies where FREE2 methylation levels, associated with FM, were elevated in different tissue types, including adult and newborn blood, lymphoblasts, chorionic villi, and primary neurons from post mortem brains 4 , 5 , 7 , 27 – 30 . Notably, the FREE2 region has also been referred to via a different name – for example FMR1 “down-stream region” as in Esanov et al .…”

Multifarious Functions of the Fragile X Mental Retardation Protein

“…A long-term question concerns roles in neurons versus glia. A recent study of human patients shows FMR1 epigenetic alterations silence FMRP specifically in neurons, but not glia or neurons obtained from reprogrammed pluripotent stem cells [13]. In the mouse FXS model, recent work shows FMRP loss changes cell differentiation kinetics for both neurons and glia (Table 1) [14], and astrocyte-specific FMRP knockout in mice increases neuronal dendritic spine density similar to the global FXS condition [15].…”

“…Partnerships with other mRNA-binding proteins could provide the long-sought specificity for coupled control of transcript targets in neuron class-specific mechanisms [40–43]. FMRP roles in glia may also contribute to FXS [13–15], and this remains an important question. Intercellular glial-neuron signaling, as well as trans -synaptic signaling, may have central roles in FXS neuropathology, which are just coming to light [63–67].…”

Targeted Reactivation of FMR1 Transcription in Fragile X Syndrome Embryonic Stem Cells

“…This finding is surprising, as previous work suggested that this methylation typically occurs coincident with or after transcriptional silencing. We explored whether this methylation might instead be by hydroxymethylation, which is not transcriptionally silencing (Brasa, Mueller et al 2016, Esanov, Andrade et al 2016). However, our preliminary studies demonstrate no evidence of hydroxymethylation in these FXS cells (data not shown).…”