Although DNA methylation is commonly found in the bodies of genes, its biological significance is unclear. Using a novel method for high-resolution analyses of the DNA methylation status of CpG islands, we identified and validated many new intragenic CpG islands that were methylated in a tissue-specific manner in normal human tissues, one of which was in SHANK3.
Through a comparative epigenomics approach, we discovered that the tissue-specific DNA methylation levels of the intragenic CpG islands of SHANK3 were evolutionarily conserved in humans and mice. In addition, the expression levels of SHANK3 were tissue-specific and evolutionarily conserved. SHANK3 encodes a structural protein in the postsynaptic densities of excitatory neurons and altered copy number, and presumably aberrant expression levels of SHANK3, underlie the abnormal neurological phenotypes in patients afflicted with 22q13 deletion syndrome and autism spectrum disorders.
The known function of SHANK3, its association with neurological diseases, and the persistence of SHANK3 tissue-specific CpG island methylation and gene expression patterns over 75 million years suggests that the intragenic DNA methylation might have a conserved function in gene expression. Because DNA methylation is known to influence the activity of promoter sequences, we searched for genetic and epigenetic evidence of in vivo promoter activity embedded within SHANK3 in mouse and human tissues. Using this integrated and cross-species approach, we identified two intragenic regions with promoter activity in vitro, are differentially methylated in vivo, and facilitate the tissue-specific transcription of novel and potentially protein-coding SHANK3 transcripts. These correlative data suggest that the intragenic methylation negatively influences promoter activity. Through a series of in vitro and in vivo DNA methylation experiments, we directly demonstrated that methylation of the intragenic promoters precluded expression.
Additionally, we found that 68.2% of intragenic sequences exhibiting tissue-specific DNA methylation identified from a genome-wide screen harbored features of promoters more commonly associated with the 5'-end of genes.
Altogether, these results support a role for intragenic DNA methylation in regulating the activity of alternate, intragenic promoters that may be generally applicable. Importantly, this study also provides insight into the transcriptional regulation of SHANK3, the level of which is critical for normal brain development.