15942
5-Hydroxymethylcytosine Is Increased in Autism Cerebellum and within the EN-2 Gene: Epigenetic Implications
Objectives: To simultaneously measure 5-mC and 5-hmC content in cerebellar tissue and within the EN-2 gene and explore the association of 5-hmC with MeCP2 binding and with 8-oxo-dG, a marker of oxidative stress and DNA damage.
Methods: In 15 case and 15 control post mortem cerebellar samples, 5-mC, 5-hmC and 8-oxo-dG were quantified by LC/MS/MS after enzymatic digestion to DNA bases and expressed per μg DNA. The EpiMark™ 5-hmC and 5-mC Analysis Kit was used to quantify 5-hmC and 5-mC within the EN-2 gene. MeCP2 binding to DNA was determined using standard chromatin immunoprecipitation methods. TET enzyme gene expression was measured using quantitative reverse transcription real-time PCR.
Results: 5-hmC and 8-oxo-dG were significantly increased relative to control cerebellar samples and demonstrated a positive correlation whereas tissue glutathione levels were significantly decreased. Unexpectedly, 5-mC levels were also significantly increased in case compared to control cerebellum and were positively correlated with 5-hmC levels. These data suggest that elevated levels of 5-hmC were stable and independent of TET 5-mC demethylation activity. Consistent with this possibility, TET2 gene expression was not significantly different between case and control cerebellum samples. Within the EN-2 promoter region, 5-hmC and 5-mC levels were also significantly increased and positively correlated. MeCP2 binding to the EN-2 promoter was significantly decreased relative to control samples.
Conclusions: Previous reports of altered DNA methylation in autism brain need to be redefined in terms of the proportion of cytosine methylation due to 5-hmC and to 5-mC. The presence of 5-hmC in cerebellum appears to be a stable epigenetic mark that is independent of TET enzyme 5-mC demethylation. Increased levels of 5-hmC may decrease gene-specific binding of MeCP2 and lead to gene overexpression. Finally, the positive correlation between 5-hmC and 8-oxo-dG suggests the possibility that oxidative stress may be a non-enzymatic mechanism to oxidize 5-mC to 5-hmC.