PROJECT SUMMARY DNA amplification is associated with pathological states such as neurological disorders, cardiac disease and cancer. At least 50% of the amplifications in cancer are transient extrachromosomal DNA (ecDNA). The transient behavior contributes to copy number plasticity, results in heterogeneous oncogene expression and alters therapeutic response. The unanswered question remains as to whether distinct mechanisms control ecDNA copy gains within cells and how they impact copy gain events associated with disease. My overall goal is to define the principles regulating selective DNA copy gains and the associated plasticity, so we may control these events. My laboratory was the first to discover a molecular basis for extrachromosomal transient site-specific DNA copy gains (TSSGs) in the human genome. Specifically, we identified the first enzyme capable of driving site-specific ecDNA amplification [the histone 3 lysine 9 and 36 tri-demethylase (H3K9/36me3) KDM4A] and demonstrated a fundamental role for epigenetic states in controlling the predilection of specific DNA regions to rereplicate and amplify. We discovered seven more chromatin enzymes- lysine methyltransferases (KMTs) and demethylases (KDMs)- that function in concert to control site-specific amplification in both non-cancer and cancer cells. These studies established a critical role for chromatin factors and their associated states in regulating DNA amplifications. With this NIGMS R35, my laboratory will expand our studies in order to elucidate: 1) the fundamental mechanisms controlling DNA amplification; 2) the molecular processes and characteristics promoting or preventing DNA amplification; and 3) the relationship between ecDNA generation and the associated RNA heterogeneity/DNA mutation burden. We will address these points by leveraging microscopy- based screens using genetic and chemical tools in order to identify key amplifiers, and in turn, generate epigenome profiles coupled to genome organization maps associated with these pathways so that molecular features controlling DNA amplification are resolved. These studies will also be coupled to state-of-the-art long read sequencing and single cell (DNA and RNA) sequencing strategies so that the associated heterogeneity within the cell population and individual cells can be correlated with the effect of the amplifier on TSSGs. These studies are being conducted in non-transformed cells that have a nearly diploid genome so that additional genomic anomalies and mutations do not impact these studies. Collectively, the data generated from these studies will increase our knowledge about the molecular features governing DNA copy gains and the associated heterogeneity, which will resolve novel biomarkers and therapeutic targets in order to control copy number- associated diseases in the years ahead.