Transcriptional and methylation profiling of adult skin keratinocytes upon aging

Currently, in-vivo epigenetic data has highlighted a plethora of methylation changes that consistently occur in the human epidermis with age (e.g. Vandiver et al, Genome Biol. 2015). These changes, however, are yet to be apportioned to either cellular ageing (e.g. free radical-induced DNA damage) or tissue structure/topology changes with age. We first plan to identify how the flattening of the epidermis with age impacts DNA methylation in keratinocytes. By seeding young keratinocyte stem cells on a young and old epidermal equivalent, we will be able to determine the effect of a flattened epidermis on its histopathology (e.g. keratinocyte differentiation rate), and the degree to which it drives epigenetic changes identified in in-vivo skin studies. Indeed, although methylation changes can take time to manifest themselves, there are large methylation changes that associate with cellular differentiation, a key process influenced by epidermal topology. Next, we will seed old keratinocyte stem cells onto identical epidermal equivalents and determine to what degree older stem cells compared to younger stem cells further recapitulate in-vivo epigenetic differences in the epidermis with age.
Once differentially methylated DNA sites are linked to topological or cellular ageing, bioinformatics techniques can be applied to identify mechanisms driving the methylation changes, such as identifying transcription factors that bind to the DNA methylation sites, and correlations between methylation and mRNA levels in publically available datasets. We will define a bioinformatic workflow for classifying and then identifying cellular and topological driven methylation changes in other epigenetic datasets. In addition, we will confirm the impact of the methylation changes using histopathological analyses of the epidermal equivalents such as confirming changes to protein levels and function.