Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum.

S J James,S Melnyk,S Shpyleva,O Pavliv,I P Pogribny

doi:10.1038/tp.2014.87

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

Highlights

Engrailed-2 (EN-2) is a developmentally regulated homeobox gene that is essential for Purkinje cell maturation and normal cerebellar development and patterning.[1]
We recently reported for the first time that alterations in DNA and histone lysine methylation within the EN-2 promoter region were associated with EN-2 overexpression in the post-mortem autism cerebellum.[2]
Using an autism candidate gene approach, we define for the first time global and gene-specific 5-hmC content in the EN-2 gene and explore the relationship between the 5-hmC level and gene expression in a chromatin context and the potential role of oxidative stress in the generation of 5-hmC

Summary

Introduction

Engrailed-2 (EN-2) is a developmentally regulated homeobox gene that is essential for Purkinje cell maturation and normal cerebellar development and patterning.[1]. EN-2 is expressed in hindbrain nuclei involved in the development of serotonin (raphe nucleus) and norepinephrine (locus coeruleus) neurotransmitter systems that have been implicated in autism.[7,8] It is important to note that the programed timing of Purkinje cell maturation and cerebellar patterning is critically dependent on perinatal EN-2 downregulation,[6,9] which is obviated by sustained EN-2 overexpression.[9,10] Our finding of sustained EN-2 gene overexpression in the postnatal autism cerebellum suggests that a critical developmental window for normal downregulation may have been missed in some individuals with autism

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Conclusion