Epigenetic patterning and maintenance are of paramount importance for normal cellular functioning and identity. Hence, pursuing the annotation of normal human tissue-specific epigenomes is an important and necessary endeavor. However, such a project is considerably more challenging than sequencing the genome because of the tissue-specific and dynamic nature of epigenomes. Thus, a more complete understanding of what constitutes a normal epigenome, and the degree to which epigenomes vary (in a tissue dependent fashion) based on aging and the environment has the potential to dramatically improve the success of studies of epigenetic alterations in disease. Hence, our work characterized methylation of phenotypically important CpG loci across several human tissue types, elucidating interindividual tissue-specific variation in methylation profiles and the contribution of CpG island context to age associated methylation alterations. This work increases our appreciation for the dynamic nature of the epigenome, and begins to define basic tenets to follow in pursuit of both constructing reference epigenomes and elucidating epigenetic alterations truly indicative of disease states.
Using recursively-partitioned mixture modeling and random forests approaches, we differentiated tissues based on CpG methylation profile, consistent with other recent studies conducting genome-wide DNA methylation profiling 
. These studies used high resolution methylation data and together have now shown that tissues have distinct methylation profiles. This novel and consistent body of work has, however, not addressed exposures in relation to interindividual variations in methylation. Not only do our findings confirm that tissue-specific epigenetic patterns can be readily defined with a targeted promoter-based CpG array, but they identify target sets of gene-loci most consistently capable of differentiating tissue types.
Factors known to contribute to methylation alterations include carcinogen exposures, inflammation, and diet. Several carcinogen exposures such as tobacco, alcohol, arsenic, and asbestos have been associated with methylation-induced gene-inactivation in various human cancers including bladder cancer, head and neck squamous cell carcinoma, and mesothelioma 
. It is therefore reasonable to suggest that various and potentially accumulating exposures throughout life may directly or indirectly lead to methylation alterations and impact disease susceptibility. Carcinogens are well known to induce genetic abnormalities that can lead to clonal selection and expansion in normal appearing tissues (termed “field effect”). Hence, the association of carcinogen exposures with the occurrence of altered methylation at phenotypically important loci may arise as a consequence of altered (“initiated”) clones. Our data suggest that large epigenetic changes occur in normal appearing tissues, and the relationship of these changes to companion genetic changes is of interest to study in the future.
Cancer is a disease of aging, and initial studies of age-related methylation in normal tissues were motivated in large part by studies of methylation in cancer 
. An early report from Issa et al.
described an association between aging colonic mucosa and estrogen receptor methylation 
. In general, trends of global (repeat element) hypomethylation and promoter hypermethylation found in cancer also have been observed in normal tissues with aging 
. In recent reports of age-related methylation in normal human prostate and colon tissues, several CpG-island-containing genes were reported to have age-related increases in methylation 
. Our results confirm these findings and, in addition, document that age-related alterations in these CpG loci are tissue-dependent. More importantly, our examination of loci with previously reported age-associated methylation alterations, in conjunction with reports from others, suggested that the relationship between aging and promoter CpG methylation is complex. For example, using restriction-landmark genome scanning of over 2000 CpG loci in T lymphocytes comparing newborns, middle age, and elderly people, Tra et al.
reported that 29 loci had age-related methylation alterations, with 23 loci displaying increased methylation with age and 6 decreasing with age 
. In addition, measuring intra-individual global methylation changes over >10 years, Bjornsson et al.
found both increased and decreased methylation levels dependent on the individual, with over 50% of participants exhibiting >5% change in methylation 
Stratifying our data on CpG-island status of loci, we showed that both the direction and strength of correlation between age and methylation were largely dependent upon CpG island status. More specifically, we found a propensity for CpG-island loci to gain methylation with age, and non-island CpGs to lose methylation with age. Our data are consistent with the literature that has demonstrated age-related increases in methylation at gene-loci found within CpG islands 
, as well as the findings of Tra et al.
and Bjornsson et al.
who showed bi-modal age-related methylation in normal tissues. A direct comparison, by examination of the data of Bjornsson et al.
, indicated that a high percentage of their top 50 most age-altered loci (all decreases in methylation) are not located in CpG islands; among 24 of 30 autosomal CpGs in their (where CpG island status can be identified by readers), only 5/24 (21%) are located in CpG islands, whereas 70% would be expected. Our results from blood samples corroborate their findings, and extend them to demonstrate similar trends in multiple other tissue types, where the strongest negative correlation between age and methylation occurs at CpGs which are not in CpG islands, and the strongest positive correlation between age and methylation occurs at loci in CpG islands.
The observed pattern of age associated methylation was irrespective of tissue-type, suggesting a common mechanism or dysregulation to explain these alterations. Reduced fidelity of maintenance methyltransferases with aging is one potential explanation for age related decreases in methylation; while age related increases in methylation could potentially reflect the accumulation of stochastic methylation events over time. As the examined tissues do not have a pathologic phenotype, methylated CpGs in these cells may not indicate dramatic functional consequences upon gene expression. However, the (in part selective) accumulation of alterations without readily detectable functional consequences should not be interpreted as biologically insignificant. Age-related drift of normal epigenomes without prominent changes in gene expression may nonetheless confer significantly increased risk of conversion to a pathologic phenotype by enhancing both the likelihood and frequency of methylation events that ultimately result in aberrant expression or altered genomic stability. For example, in the context of acquired “non-functional” CpG methylation in the promoter region of an aged individual, continued stochastic methylation events (e.g. “methylation spreading”) increase the chance of methylation induced silencing at that promoter (or silencing of another locus through action at a distance via silencing of other important regions such as enhancers), and hence, progression to a pathologic phenotype. Certainly, this hypothesis is especially plausible for the many diseases of aging. Alternatively, aberrant CpG methylation that silences a gene on a single allele may not appear to have a functional consequence if the complementary allele can provide compensatory expression. As a result, for example, clusters of cellular clones with mono-allelic gene expression could contribute to an increased risk of progression to a pathologic phenotype (e.g. loss of the 2nd functional allele). Future population-based studies addressing the potential of quantifying age and/or exposure associated methylation alterations indicative of disease risk are necessary.
We have provided clear evidence of interindividual variation in tissue–specific methylation related to aging and environmental exposures at disease-relevant CpGs across 10 normal human tissue types. We have demonstrated both general and tissue specific alterations, uncovered a CpG island context-dependent directionality to age associated methylation alterations, and provided a novel path for examining the mechanistic basis of these alterations. By enumerating the methylation status of a panel of cancer-related genes known to stably control transcription in normal tissues, we have also afforded important controls for comparison to diseased tissues, potentially aiding in identification of the most critical alterations in specific diseases and providing more robust targets for novel treatments. Importantly, we have also begun to disentangle the contributions of aging and environmental factors to methylation alterations in normal tissues. Uncovering age and exposure-related methylation changes and their clear contextual dependence is an important contribution to our basic understanding of epigenetic maintenance as it relates to both aging and the pathologic process, provides a potential avenue to pursue clinically useful biomarkers, as well as to identify novel markers of disease susceptibility.