A large number of individual CpG sites were shown to be strongly associated with chronological age and DNA methylation levels. A potential confound of note is the dynamic cellular composition of human brain, particularly that the proportion of neurons to glia may change with chronological aging. Because we have identified consistent results across multiple brain regions, this is not likely to be a confound among the 10 CpG sites that showed significant genome-wide association with chronological age across all four brain regions. Of the 10 loci identified, one locus within the MYOD1 gene has previously been reported to be associated with age-related methylation changes in the brain and the pleura (7
). In addition, 3 of the 10 loci identified were also shown to be associated with age in human blood (10
). Collectively, these data support the notion that the age-associated CpG sites identified in our data are not an artifact of age-associated alterations in cellularity, but rather reflect an underlying biological change in DNA methylation status.
The consistent findings from our group and others showing that the patterns of both DNA methylation and gene expression are quite different in cerebellum compared with other brain tissues (6
) may be attributed to the unique nuclei of cerebellar purkinje neurons, which are large, euchromatic structures that exhibit a greater proportion of 5-hydroxymethylcystosine modifications compared with other neuronal populations (12
). Although these factors alone may not account for the substantive divergence observed in our current analyses, they do illustrate that the genetic component of this tissue exhibits tangible differences from that within other brain regions. Thus, it is not surprising that age-related DNA methylation sites would be most divergent in cerebellar tissue compared with the other brain regions tested here.
The classes of genes identified at age-associated sites included DNA-binding factors and transcription factors, illustrating a strong enrichment for genes related to DNA binding, morphogenesis and regulation of transcription. Given the functional nature of these clusters, it is conceivable that altered epigenetic regulation at these loci may give rise to quite broad changes in transcriptional potential during the aging process. The clustering of age-associated CpG methylation sites that are proximal to genes associated with DNA binding could have several biological implications. Given that the genes in the DAVID clusters (Table ) are not associated with DNA damage argues against these associations being a response to pathological events such as reactive oxygen species-mediated damage of nucleic acids. Rather, these genes are responsible for transcription with the homeobox proteins being especially prominent. This suggests that age-related alterations in methylation might be important for the maintenance of transcriptional programs in aging tissues. In this context, it is of interest that there are relatively few mRNA changes that show linear association with aging, although there is a tendency for gene expression to show higher variance as organisms age (13
). An interesting possibility therefore, is that the accumulation of DNA methylation may be important in maintenance of consistent gene expression patterns with age.
Here, we describe CpG sites that exhibit strong age-associated changes in DNA methylation. We see a large number of statistically significant age-associated changes in DNA methylation, despite a conservative correction for multiple testing. Many of these CpG sites were significant in multiple tissues and occur at higher frequencies than one would expect by chance. We saw a highly significant enrichment of age-associated methylation changes at CpG islands of functionally related transcripts. Finally, the majority of such associations were positive, showing that methylation tends to increase with age.
We observed an excess of shared age-associated CpG sites across more than one of the four selected brain tissues, suggesting that altered cellular composition was not the underlying cause of changing the DNA methylation profile, but rather that there was a common regulatory mechanism across the brain regions. The classes of genes identified at the age-associated sites included DNA-binding factors and transcription factors; therefore, one might surmise that the age-associated changes in methylation are likely to be associated with the maintenance of transcriptional programs.
In conclusion, we present a comprehensive analysis of DNA methylation across the four distinct human brain tissues. Our data suggest that there are specific loci where the DNA methylation level changes with the chronological age in the human brain and underscores the necessity to study DNA methylation in aging research in order to understand the underlying mechanism and its functional effects.