title: Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum.
abstract: Epigenetic mechanisms regulate programmed gene expression during prenatal neurogenesis and serve as a mediator between genetics and environment in postnatal life. The recent discovery of 5-hydroxymethylcytosine (5-hmC), with highest concentration in the brain, has added a new dimension to epigenetic regulation of neurogenesis and the development of complex behavior disorders. Here, we take a candidate gene approach to define the role 5-hmC in Engrailed-2 (EN-2) gene expression in the autism cerebellum. The EN-2 homeobox transcription factor, previously implicated in autism, is essential for normal cerebellar patterning and development. We previously reported EN-2 overexpression associated with promoter DNA hypermethylation in the autism cerebellum but because traditional DNA methylation methodology cannot distinguish 5-methylcytosine (5-mC) from 5-hmC, we now extend our investigation by quantifying global and gene-specific 5-mC and 5-hmC. Globally, 5-hmC was significantly increased in the autism cerebellum and accompanied by increases in the expression of de novo methyltransferases DNMT3A and DNMT3B, ten-eleven translocase genes TET1 and TET3, and in 8-oxo-deoxyguanosine (8-oxo-dG) content, a marker of oxidative DNA damage. Within the EN-2 promoter, there was a significant positive correlation between 5-hmC content and EN-2 gene expression. Based on reports of reduced MeCP2 affinity for 5-hmC, MeCP2 binding studies in the EN-2 promoter revealed a significant decrease in repressive MeCP2 binding that may contribute to the aberrant overexpression of EN-2. Because normal cerebellar development depends on perinatal EN-2 downregulation, the sustained postnatal overexpression suggests that a critical window of cerebellar development may have been missed in some individuals with autism with downstream developmental consequences. Epigenetic regulation of the programmed on-off switches in gene expression that occur at birth and during early brain development warrants further investigation.
authors.affiliation: Department of Pediatrics, University of Arkansas for Medical Sciences, Arkansas Children's Hospital Research Institute, Little Rock, AR, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: James
authors.affiliation: Department of Pediatrics, University of Arkansas for Medical Sciences, Arkansas Children's Hospital Research Institute, Little Rock, AR, USA.
authors.forename: S
authors.initials: S
authors.lastname: Shpyleva
authors.affiliation: Department of Pediatrics, University of Arkansas for Medical Sciences, Arkansas Children's Hospital Research Institute, Little Rock, AR, USA.
authors.forename: S
authors.initials: S
authors.lastname: Melnyk
authors.affiliation: Department of Pediatrics, University of Arkansas for Medical Sciences, Arkansas Children's Hospital Research Institute, Little Rock, AR, USA.
authors.forename: O
authors.initials: O
authors.lastname: Pavliv
authors.affiliation: Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR, USA.
authors.forename: I P
authors.initials: IP
authors.lastname: Pogribny
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000293:Adolescent; D001321:Autistic Disorder / Q000235:genetics* / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism*; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D005260:Female; D015870:Gene Expression / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D008297:Male; D051783:Methyl-CpG-Binding Protein 2 / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D000099266:Engrailed 2 Protein
chemical_list: D044503:5-Methylcytosine; D003596:Cytosine; D018398:Homeodomain Proteins; D051783:Methyl-CpG-Binding Protein 2; D009419:Nerve Tissue Proteins; D000099266:Engrailed 2 Protein; C011865:5-hydroxymethylcytosine