ABSTRACT The incidence of cardiovascular disease (CVD), Type 2 diabetes (T2D) and cancers all dramatically increase as a function of age. The underlying mechanisms of these diseases, which vary, are incompletely understood. Genome-wide association studies (GWAS) have identified many SNPs that are associated with these conditions. One of the strongest associations comes from the CDKN2A/B locus on chromosome 9p21.3 which has been associated with multiple age-related diseases, as well as overall human lifespan. Within this 200 kb locus, there are three encoded proteins, p16INK4a, p14ARF and p15INK4b, and one antisense non-coding RNA, the inhibitor of CDK4 (INK4) locus (AS/ANRIL). To date, it has not been firmly established which, if any, of these genes are the risk genes for the associated diseases. There are ~193 disease-associated, noncoding SNPs in linkage disequilibrium (LDs) across this 200 kb region, represented by 18 lead SNPs used for GWAS analysis. While the mechanisms underlying the contribution of these SNPs to specific diseases are not fully understood, a single genetic region associated with multiple different age-related diseases suggests that this locus may modulate these conditions by promoting aging itself, perhaps via induction of cellular senescence as a common mechanism. In this application, we propose to apply an experimental approach using high throughput techniques we have recently developed including Reel-seq and FREP/SDCP-MS, to systematically dissect this locus. We will first identify the disease-associated functional SNPs (fSNPs), as well as the regulatory elements across the 58 kb core region primarily associated with cardiovascular diseases using Reel-seq. Next, we will identify the regulatory proteins that specifically bind to all the fSNPs, as well as the regulatory elements, using FREP/SDCP- MS. A range of relevant cell types related to atherosclerosis will be used to generate the nuclear extract required for our screens. We will demonstrate the role of these regulatory proteins by confirming their direct effects on p16INK4a, p14ARF, p15INK4b and AS/ANRIL expression, and subsequently on cell cycle regulation and cellular senescence. A range of complementary techniques such as RNAi, CRISPR/cas9 gene editing, will be employed. Such analysis will provide the first in-depth understanding of this critical genomic region, as well as a unique strategy to uncover unifying biochemical pathways that simultaneously regulate atherosclerosis, as well as potentially multiple other age-related diseases.