Complex Epigenetic Regulation of Engrailed-2 (EN-2) Homeobox Transcription Factor Gene in the Autism Cerebellum

Background:  The EN-2 homeobox gene is considered to be an autism susceptibility gene based on neuroanatomical parallels between autism and cerebellar developmental abnormalities in rodent models and also on family linkage studies indicating an over-transmission of EN-2 polymorphic variants from parents to affected children.  The normal timing of Purkinje cell maturation and cerebellar patterning is critically dependent on perinatal EN-2 down-regulation, which is disrupted with EN-2 overexpression.

Objectives:  The elucidation of epigenetic alterations in the autism brain has the potential to provide new insights into the molecular mechanisms underlying abnormal gene expression associated with this disorder.  Given strong evidence that EN-2 is a developmentally expressed gene relevant to cerebellar abnormalities and autism, the epigenetic evaluation of this gene was undertaken.

Methods:  Frozen cerebellar samples from 26 case and control post mortem cerebellar samples were matched for age, gender, PMI, race, and cause of death.  Assessments included genome-wide DNA methylation, EN-2 promoter methylation, EN-2 gene expression and protein levels.  In addition, chromatin immunoprecipitation methodology was used to evaluate trimethylation status of histone H3 lysine 4 (H3K4) associated with gene over-expression and H3 lysine 27 (H3K27) associated with gene down-regulation. The binding of GATA-1, an enhancer element with binding motifs in the EN-2 promoter, was also evaluated.  

Results:  The results revealed an unusual pattern of global and EN-2 promoter DNA hypermethylation that was accompanied by an increase in EN-2 gene expression and protein levels. Consistent with EN-2 over-expression, histone H3K27 trimethylation mark in the EN-2 promoter was significantly decreased in the autism samples relative to matched controls (p=0.02).  Supporting H3K27 demethylation and increased EN-2 gene expression, mean level of H3K4 trimethylation was found to be increased and the binding of GATA-1 enhancer element was decreased.  The unexpected gene and protein over-expression in the presence of promoter DNA hypermethylation may be partially explained by over-riding histone methylation patterns in H3K27 and H3K4.

Conclusions:   The epigenetic evaluation of EN-2 in the autism cerebellum herein indicates a persistent up-regulation of this developmentally expressed homobox gene that normally undergoes perinatal down-regulation to insure normal timing and onset of Purkinje cell differentiation.  Together, the results suggest that normal EN-2 down-regulation that signals Purkinje cell maturation during late prenatal and early post-natal development may not have occurred in some individuals with autism and that the postnatal persistence of EN-2 overexpression may contribute to cerebellar abnormalities in these individuals.