Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum

Abstract

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by symptoms related to altered social interactions/communication and restricted and repetitive behaviors. In addition to genetic risk, epigenetic mechanisms (which include DNA methylation/demethylation) are thought to be important in the etiopathogenesis of ASD. We studied epigenetic mechanisms underlying the transcriptional regulation of candidate genes in cerebella of ASD patients, including the binding of MeCP2 (methyl CpG binding protein-2) to the glutamic acid decarboxylase 67 (GAD1), glutamic acid decarboxylase 65 (GAD2), and Reelin (RELN) promoters and gene bodies. Moreover, we performed methyl DNA immunoprecipitation (MeDIP) and hydroxymethyl DNA immunoprecipitation (hMeDIP) to measure total 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in the same regions of these genes. The enrichment of 5-hmC and decrease in 5-mC at the GAD1 or RELN promoters detected by 5-hmC and 5-mC antibodies was confirmed by Tet-assisted bisulfite (TAB) pyrosequencing. The results showed a marked and significant increase in MeCP2 binding to the promoter regions of GAD1 and RELN, but not to the corresponding gene body regions in cerebellar cortex of ASD patients. Moreover, we detected a significant increase in TET1 expression and an enrichment in the level of 5-hmC, but not 5-mC, at the promoters of GAD1 and RELN in ASD when compared with CON. Moreover, there was increased TET1 binding to these promoter regions. These data are consistent with the hypothesis that an increase of 5-hmC (relative to 5-mC) at specific gene domains enhances the binding of MeCP2 to 5-hmC and reduces expression of the corresponding target genes in ASD cerebella.

Semiconductor-based sequencing of genome-wide DNA methylation states

“…54,57 Our MeDIP-Seq data from human brain reveal the mutual exclusivity of these DNA modifications and provides additional support for a role of 5hmC distinct from that of 5mC, including a previously unappreciated potential role at the TES of genes. Future studies that utilize 5hmC MeDIP-Seq as a cost-effective and rapid technique to assess differences between the brain from normal and disease conditions, such as Autism Spectrum Disorder, 61 Alzheimer disease, 62 or Schizophrenia, 63 may lead to further insight into the functional importance of 5hmC. Given the appropriate antibodies, this Ion Torrent MeDIP-Seq approach potentially offers a means to study the functional importance of other poorly understood DNA modification variants such as 5-formylcytosine and 5-carboxycytosine.…”

5-Hydroxymethylcytosine, the “Sixth Base”, during brain development and ageing

“…In the context of Alzheimer's disease there is increasing evidence that changes of 5hmC play a crucial role for the progression of the disease (Bradley-Whitman and Lovell 2013;Condliffe et al 2014;van den Hove et al 2012). But also in the context of mental disorders, recent studies imply that 5hmC is closely associated with autism (James et al 2014;Zhubi et al 2014).…”

Epigenetic Regulations of GABAergic Neurotransmission: Relevance for Neurological Disorders and Epigenetic Therapy

“…However, other studies challenge this finding by showing no significant reduction in GABRB3 mRNA and protein levels in brain tissues of MeCP2-deficient mice [114,115]. LaSalle reasons the time point of MeCP2-deficient mice used or RT-PCR issues to explain the discrepancies observed in these later studies [105,106,116]. Some studies have linked the decreased GABRB3 expression in MeCP2 deficiency with decreased expression of the ubiquitin protein ligase E3A (UBE3A) gene [109,112,113].…”

“…One of the above studies that looked at cytoplasmic adaptor protein Disabled-1 (Dab1) KO mice showed no reelin-mediated phosphorylation and calcium influx demonstrating that phosphorylation of NMDAR receptor is a key step of the mechanism; Dab1 acts downstream of reelin to mediate the phosphorylation [98]. Another important player in the DNMT-regulated hypermethylation is MeCP2, an epigenetic reader, which shows increased affinity to methylated CpG in GAD67 and RELN gene promoters and acts as a transcriptional repressor [104,105,106]. MeCP2 and HDAC are the components of the repressor complex formed in the DNMT1-methylated site, which is seen in SZ patients or SZ mice models [107].…”