RetroDecoys: Temporally-regulated and cell type selective technology for transcriptional control

项目摘要/摘要 转录因子在细胞中协调最动态的过程-从产生级联的抗病毒和 抗生素耐药蛋白对分化和转移的影响。即使是一个单一物种的丰度的变化 转录因子可以诱导基因表达模式，从根本上改变转录组和 细胞生理学细胞的生理学有趣的是，转录因子对基因表达的影响是 因子本身的丰度以及其在DNA中结合部位的丰度。作为数量的 DNA结合部位增多，各转录因子分子的作用减弱。然而，在细胞中，蛋白质 丰度的变化和DNA结合位点的数量是静止的，所以转录因子的量是唯一的 动态控制点。我们的目标是设计和测试一种分子技术，这种技术将产生丰富的 体内DNA中的转录因子结合位点，按需提供。这些DNA分子将争夺占有率 通过转录因子，从而拮抗它们对内源基因的作用。为了建造这项技术， 我们将在可诱导和细胞类型的控制下，设计逆转录元件来逆转录靶部位 特定的推动者。这些元素可以作为可编程部件集成到遗传电路中，以合成 生物学和一种对转录组施加控制的方法，转录组对细胞的状态做出反应。它们还可以使 转录因子的拮抗作用目前还没有小分子拮抗剂。我们将对此进行验证 方法在两个临床相关的模型：癌症进展和抗生素耐药性。

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.