Complex epigenetic regulation of Engrailed-2 (EN-2) homeobox gene in the autism cerebellum

S J James,Stepan Melnyk,Svitlana Shpyleva,I P Pogribny,Oleksandra Pavliv

doi:10.1038/tp.2013.8

Abstract

The elucidation of epigenetic alterations in the autism brain has potential to provide new insights into the molecular mechanisms underlying abnormal gene expression in this disorder. Given strong evidence that engrailed-2 (EN-2) is a developmentally expressed gene relevant to cerebellar abnormalities and autism, the epigenetic evaluation of this candidate gene was undertaken in 26 case and control post-mortem cerebellar samples. Assessments included global DNA methylation, EN-2 promoter methylation, EN-2 gene expression and EN-2 protein levels. Chromatin immunoprecipitation was used to evaluate trimethylation status of histone H3 lysine 27 (H3K27) associated with gene downregulation and histone H3 lysine 4 (H3K4) associated with gene activation. The results revealed an unusual pattern of global and EN-2 promoter region DNA hypermethylation accompanied by significant increases in EN-2 gene expression and protein levels. Consistent with EN-2 overexpression, histone H3K27 trimethylation mark in the EN-2 promoter was significantly decreased in the autism samples relative to matched controls. Supporting a link between reduced histone H3K27 trimethylation and increased EN-2 gene expression, the mean level of histone H3K4 trimethylation was elevated in the autism cerebellar samples. Together, these results suggest that the normal EN-2 downregulation that signals Purkinje cell maturation during late prenatal and early-postnatal development may not have occurred in some individuals with autism and that the postnatal persistence of EN-2 overexpression may contribute to autism cerebellar abnormalities.

Highlights

Supporting a link between reduced histone H3K27 trimethylation and increased EN-2 gene expression, the mean level of histone H3K4 trimethylation was elevated in the autism cerebellar samples. These results suggest that the normal EN-2 downregulation that signals Purkinje cell maturation during late prenatal and early-postnatal development may not have occurred in some individuals with autism and that the postnatal persistence of EN-2 overexpression may contribute to autism cerebellar abnormalities
Engrailed-2 (EN-2) is considered to be an autism susceptibility gene based on neuroanatomical parallels between autism and cerebellar developmental abnormalities in rodent models, and on family linkage studies indicating an overtransmission of EN-2 polymorphic variants from parents to affected children.[1]
Normal timing of Purkinje cell maturation and cerebellar patterning is critically dependent on perinatal EN-2 downregulation,[2] which is disrupted with EN-2 overexpression.[4]

Summary

Introduction

Engrailed-2 (EN-2) is considered to be an autism susceptibility gene based on neuroanatomical parallels between autism and cerebellar developmental abnormalities in rodent models, and on family linkage studies indicating an overtransmission of EN-2 polymorphic variants from parents to affected children.[1] In mice, EN-2 is highly expressed in Purkinje cells during fetal and early-postnatal development acting primarily as a transcriptional repressor until it is downregulated during the perinatal period.[2] 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.[3] Importantly, normal timing of Purkinje cell maturation and cerebellar patterning is critically dependent on perinatal EN-2 downregulation,[2] which is disrupted with EN-2 overexpression.[4] human studies are limited, expression analysis of 18- to -21-week-old fetuses indicated widespread EN-2 gene expression throughout the mid-/hindbrain regions including the cerebellar cortex and deep nuclei at 40-week gestation.[5,6] The human EN-2 gene is composed of two exons separated by a single intron and spans only 8 kb of DNA mapping to 7q36.3, just distal to a region that has been reproducibly associated with autism in linkage studies.[7,8]

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Conclusion