Progressive DNA Hypomethylation as a Measure of Mitotic History and Potential Contributor to Replicative Senescence.

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PROJECT SUMMARY / ABSTRACT Cellular and molecular alterations that accumulate during cell division are considered to be contributors to aging phenotypes. Changes in epigenetic marks, including DNA methylation, have been widely documented in aging. This has spurred the development of epigenetic molecular clocks, which rely primarily on replication- independent gain of methylation at CpG islands. As molecular clocks tuned to chronological time across different tissue types with varying mitotic rates and histories, these clocks are not directly linked to mitotic cell division. In contrast, we have found that loss of DNA methylation at lamina-attached, late-replicating regions of the genome is closely tied to apparent mitotic history. We propose to use primary human cell culture to experimentally validate that mitosis is a driver of hypomethylation, and to explore the underlying mechanisms and consequences of this hypomethylation, to define genomic and chromatin features driving hypomethylation, and use this data to construct a mitotic molecular clock. In Specific Aim 1 we propose to use primary human cell culture to provide experimental evidence for the contribution of mitosis to PMD hypomethylation, and to disentangle the time-dependency and mitosis- dependency of the phenomenon using cell cycle inhibition. We will also investigate whether enhancing maintenance methylation machinery is able to counteract the progressive loss of DNA methylation. In Specific Aim 2 we propose to investigate whether DNA hypomethylation contributes to replicative senescence or associated phenotypes in primary human cell culture. We will investigate whether inhibition of maintenance methylation accelerates senescence, and whether progressive hypomethylation is extended in telomerase- immortalized primary human fibroblasts, contributing to premalignant phenotypes associated with such immortalization. We will also investigate whether accelerated senescence by progerin expression or supra- physiologic O2 leads to accelerated DNA hypomethylation. In Specific Aim 3 we will characterize the genomic and chromatin features that influence individual CpG hypomethylation rates. We will measure the rates of DNA hypomethylation of individual CpGs in six different primary human cell types and use this information to identify cell-type-specific, as well as universal genomic and chromatin drivers of hypomethylation. Finally, we will use elastic net regression on the assembled data to construct cell-type-specific and universal epigenetic mitotic clocks in Specific Aim 4. The preliminary studies strongly support the concept and feasibility of a DNA hypomethylation-based mitotic clock. The outcome of this proposed research could have important impacts on our understanding of the contribution of widespread hypomethylation to aging phenotypes, with potential implications for aging interventions. The availability of accurate molecular clocks specifically designed to measure mitotic history would provide a valuable molecular tool to characterize cells and tissues in human health and disease.