PROJECT SUMMARY / ABSTRACT Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by the rapid onset of premature aging beginning in childhood. The condition is caused by a mutation in the nuclear lamin LMNA gene, which leads to the production of a dominant gain-of-function isoform of the protein, called progerin. Progerin causes major disruptions to nuclear morphology and function, including nuclear protein export and mitosis, replication stress, and contributes to increased DNA damage as well as the loss of heterochromatin and dysregylation of other epigenetic marks, including cytosine-5 DNA methylation. We have recently provided direct experimental evidence that aging-associated loss of DNA methylation at nuclear lamina-attached regions of the genome is a direct consequence of cell division-associated DNA replication. We used this principle to develop an epigenetic mitotic clock, called RepliTali, which provides a reliable estimate of cellular replicative history. We hypothesize that altered DNA methylation patterns in patients with HGPS may contribute actively to the severely accelerated aging observed in patients with HGPS. Here, we propose to conduct high- resolution analyses of DNA methylation alterations in serially cultured HGPS cells and to investigate whether we can extend the lifespan of HGPS model systems by manipulating DNA methylation patterns. In Specific Aim 1 we will define DNA methylation dynamics in serially cultured HGPS fibroblasts from early passage through replicative senescence using cost-effective Infinium DNA methylation arrays. In Specific Aim 2 we will conduct high-resolution single-cell methylome analyses at key stages of HGPS fibroblast culture to detect arising aberrations and delineate population transitions. We have developed a single-cell whole-genome bisulfite sequencing (sc-WGBS) method that delivers genomic coverage far superior to any other published sc-WGBS methods. In Specific Aim 3 we will investigate whether DNA methylation manipulation can extend lifespan in HGPS models. In Aim 3A we will test whether overexpression of DNA methylation writers and erasers increases the replicative lifespan of HGPS fibroblasts. In Aim 3B, we will target DNA methyltransferase overexpression to arterial smooth muscle cells in an HGPS mouse model to investigate whether this reduces arterial smooth muscle loss and extends lifespan. We will monitor DNA methylation changes in this mouse model using a new cost-effective DNA methylation array. We present extensive and compelling preliminary data that demonstrates both the feasibility and relevance of the proposed aims. The outcome of this proposed research could have important impacts on our understanding of the contribution of DNA methylation alterations to HGPS phenotypes, potentially opening avenues for new therapeutic approaches to treat progeria. In addition, this study could shed light on similar mechanisms operating at a longer timescale ...