Alterations of Epigenetic Mechanisms in post-mortem Autism Spectrum Disorder (ASD) subjects

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by impaired social communication and social interactions as well as repetitive and restrictive behaviors. Recently, there has been increase interest in studying epigenetic mechanisms operative in ASD etiopathogenesis. These mechanisms involve DNA methylation and histone modifications. Several lines of evidence implicate cerebellum as one of the brain areas involved in the pathology of ASD. Furthermore, postmortem cerebellar studies have consistently reported abnormalities in the expression of genes associated with GABAergic neurons, such as glutamic acid decarboxylase 67 (GAD1) and 65 (GAD2), as well as genes associated with glutamatergic neurons, such as Reelin (RELN). This project focuses on studying epigenetic mechanisms implicated in the transcriptional regulation of the candidate genes in cerebella of ASD, including the binding of Methyl-CpG binding protein 2 (MeCP2), DNA-methyltransferase 1(DNMT1) and Ten eleven translocator 1(TET 1) to the promoters and gene bodies of GAD1, GAD2 and RELN. For quantification of methylation (5-mC enrichment) and hydroxymethylation (5-hmC enrichment) status of these genes, we performed methyl DNA immunoprecipitation (MeDIP) and hydroxymethyl DNA immunoprecipitation (hMeDIP) assays. Lastly, Tet assisted bisulfite (TAB) pyrosequencing was utilized to validate findings obtained by MeDIP and hMeDIP measurements. The results showed increased binding of MeCP2 and TET1 to the promoter regions of GAD1 and RELN, but not to the corresponding gene bodies in cerebellar cortex of ASD. In contrast, DNMT1 expression and the level of binding to the promoters or gene body regions of corresponding genes did not change in ASD compared to CON. Finally, we detected enrichment in the level of 5-hmC, but not 5-mC, at the promoters of GAD1 and RELN in ASD when compared with CON. In conclusion, increased expression and binding of TET1 to the promoters of GAD1 and RELN facilitates 5-hmC enrichment, which enhances binding of MeCP2 to the same gene domains and ultimately results in reduced expression of the corresponding target genes in ASD cerebella.