DNA Methylation and Cell Type Specification
DNA methylation plays a central role in embryogenesis, and this importance continues throughout development  . In the immune system, for example, maintenance of DNA methylation patterns is required for hematopoietic stem cell self-renewal and differentiation . During hematopoietic stem cell differentiation, DNA methylation gains and losses at specific regions of the genome “lock-in” differentiation marks that allow cells with the same genetic material to express only the specific genes required for their unique cellular processes and identities . This is illustrated by the lineage- specific epigenetic differences that arise when early multipotent progenitors split into myeloerythroid and lymphoid lineages; these patterns become more specific as differentiation progresses, resulting in cell type specific DNA methylation signatures in mature cells [24-28] (Fig. 3.2] . Hematopoietic stem cells lacking the maintenance methyltransferase prematurely lose their self-renewal capabilities, and cells lacking de novo methyltransferases show impaired lineage commitment, illustrating that these DNA methylation changes are essential for lineage development [29, 30].
Because of these cell type specific DNA methylation patterns, cell and tissue types are the largest determinants of DNA methylation variation in healthy individuals [31-33] . It is important to note that these differences in cell type DNA methylation patterns can create at least two major challenges for studies examining the role of DNA methylation in organs or tissues composed of multiple cell types: inter-individual variation in cell types and the concordance between central and surrogate tissues. First, inter-individual cell type differences within a tissue can induce confounding effects that may mask or overwhelm another biological signal with a smaller effect size. In blood, for example, it is essential to control for differences in white blood cell composition between individuals. When cell count information is not available, DNA methylation patterns can be used to predict the underlying cellular composition in order to control for it [28, 34]. This tool is particularly important when studying the relationship between DNA methylation and age in blood tissue, as it has been shown that white blood cell composition changes drastically with age, and that failing to control for these changes can result in white blood cell DNA methylation lineage markers being mistaken for age-associated DNA meth- ylation sites . In regards to tissues other than blood, similar predictive models exist for neurons versus glia in the brain, and other methods exist that can control for cell type differences without specifically predicting underlying cell composition
Fig. 3.2 Representation of changes in DNA methylation during hematopoietic stem cell differentiation. Gain and loss of DNA methylation at lineage-specific genomic regions confers cell-type specificity. Methylation patterns become more unique as differentiation progresses. The shaded boxes represent DNA methylation at specific regions of the genome. For example, the black- shaded box in the hematopoietic stem cell represents DNA methylation levels at genes that enable pluripotency. This mark is slowly lost as the cell becomes more differentiated, and marks of specific cell types arise [36-38]. The second challenge presented by cell type specific DNA methylation patterns arises when studying surrogate tissues. In human studies, tissues of interest are often inaccessible or require invasive collection methods. To address this, easily collected surrogate tissues, such as blood or cheek swabs, are substituted. However, given the tissue specificity of DNA methylation, it can be challenging to make biological interpretations of function in the tissue of interest when using these alternatives. Ongoing research into the concordance of DNA methylation between tissues collected post-mortem, such as brain and blood, is making the study of surrogate tissues increasingly more interpretable and valuable [33, 39, 40].