Project Summary Site-specific intra-chromosomal DNA amplification is common in many cancers, but the events that initiate this process are unknown. To address this question, we will use one of only two known systems where this occurs as a normal process in development, namely in the salivary gland polytene chromosome DNA puffs of the fly Sciara. We propose to elucidate the mechanism for induction of DNA puff amplification, making use of our recent advances in development of a method for Sciara transformation and assembly of the Sciara genome. We propose a model where an upstream enhancer (DHS = DNase hypersensitive site) specifies the location of an origin through a looping interaction. We posit that developmental recruitment of an amplification factor (such as EcR = ecdysone receptor) causes origin re-replication resulting in DNA amplification. To test this model, first, we will map the origins of replication and re-replication in the salivary gland genome by SNS-seq and OK-seq. This will identify any consensus motifs that map uniquely at amplification origins (such as EcREs at the origin). 4C will assay if there is a looping interaction between the DHS and the origin. Mutagenesis will test if cis-elements (e.g., DHS, origin EcREs) have functional roles for origin specification and re-replication. Next, we will identify the trans-acting factors (protein and RNA) that bind to the cis-elements (DHS and origin) by co- immunoprecipitation, CUT&RUN to assay sites of in vivo occupancy by EcR, and proximity labeling by a fusion of APEX2 with EcR or ORC2 (origin recognition complex) to identify by mass spectrometry (LC-MS/MS) the proteins that interact with them. Candidate proteins that associate with the DHS and/or origin will be knocked- down to assay if they are required for DNA amplification. These results will be confirmed and extended in an unbiased approach using CRISPR targeting of APEX2 to the DHS or origin for proximity labeling to identify proteins bound at these sites. Similar directed and unbiased approaches will identify and test the function of ncRNA at the DHS and/or origin. Together, these experiments will test the model that the DHS is important for origin specification and if it does so by a looping interaction. Factors (proteins and ncRNA) that mediate this interaction will be identified. Many of these are expected to be present at normal replication. The experiments should identify the developmentally regulated factor that joins this protein complex and triggers re-replication. For example, EcR might interact with components of the pre-replication complex to allow continual loading and activation of Mcms. Our results will suggest a mechanism for DNA puff amplification, and future experiments can test if this may serve as a paradigm for initiation of DNA amplification in cancer.