Project Summary/Abstract Transcription factors orchestrate the most dynamic processes in cells – from producing cascades of antiviral and antibiotic resistance proteins to differentiation and metastasis. A change in the abundance of even a single transcription factor can induce a pattern of gene expression that fundamentally alters the transcriptome and physiology of a cell. Interestingly, the effect that a transcription factor has on gene expression is a function of both the abundance of the factor itself as well as the abundance of its binding site in DNA. As the number of DNA binding sites increases, the effect of each transcription factor molecule decreases. Yet, in cells, protein abundance changes and the number of DNA binding sites is static, so the transcription factor amount is the only point of dynamic control. We aim to engineer and test a molecular technology that will produce abundant transcription factor binding sites in DNA in vivo, on demand. These DNA molecules will compete for occupancy by the transcription factors, thereby antagonizing their action on endogenous genes. To build this technology, we will engineer retroelements to reverse transcribe the target site under the control of inducible and cell-type specific promoters. These elements can be integrated into genetic circuits as programmable parts for synthetic biology and a means to exert control over a transcriptome that is responsive to a cell's state. They also enable antagonism of transcription factors that currently have no small molecule antagonist. We will validate this approach in two clinically relevant models: cancer progression and antibiotic resistance.