Targeted Gene Regulation Using Engineered Synthetic Transcriptional Regulators

项目摘要 转录因子是关键的功能蛋白，通过与基因的直接相互作用来调节基因的表达 基因组DNA的特定序列，从而激活或抑制转录。这一高度保守的 过程控制许多中央生物学功能，包括细胞内稳态、分化和凋亡。 鉴于这种在调节细胞状态和功能中的核心作用，异常活动被牵连到 许多疾病，包括神经紊乱、自身免疫、糖尿病和癌症。尤其是，大师 调控因子MYC转录因子在超过一半的癌症中失控，但有效的靶向治疗 由MYC驱动的基因程序尚未开发出来。而涉及直接针对MYC的方法 已经探索了小分子和抗体或间接靶向，如基因沉默，这些是 与主要缺陷有关，包括结合作用或活性弱，药理作用差 属性和偏离目标的效果。作为另一种方法，建模实验室最近开发了一种新的课程 从转录因子MAX衍生的超稳定合成DNA结合域的正交性自我- 二聚体以序列特异性结合DNA，具有高亲和力和特异性。这些人工合成的转录 调节剂(STR)竞争性地抑制MYC/MAX或MAX/MAX和CORT的天然二聚体的DNA结合 MYC依赖的基因表达。在这一先例的基础上，我假设这些 化合物将通过共价连接分裂的单体STR来增加，以产生预先组织的二聚体 STR。此外，我假设，产生多功能的STR可以招募转录机器来 定向的基因组位置将提供进一步的控制，以抑制MYC依赖的表型。建议数 研究旨在探索这两条推进STR技术的潜在途径，并评估其效果 MYC依赖的基因表达和表型。结合起来，这种串联的方法将建立一个新的阶层 并为研究细胞中致癌基因的表达提供了机制工具。 此外，这种开发转录因子模拟的模块化设计策略将提供一个框架，以 能够为未来针对其他重要转录因子和基因组量身定做的构建物做准备 目标。

PROJECT ABSTRACT Transcription factors are key functional proteins that regulate gene expression through direct interaction with specific sequences of genomic DNA, thereby activating or repressing transcription. This highly conserved process controls many central biological functions including cellular homeostasis, differentiation, and apoptosis. Given this central role in regulating cell state and function, it is unsurprising that aberrant activity is implicated in many diseases including neurological disorders, autoimmunity, diabetes, and cancer. In particular, the master regulator MYC transcription factor is dysregulated in over half of all cancers, yet effective therapies targeting MYC-driven gene programs have yet to be developed. While approaches involving direct targeting of MYC with small molecules and antibodies or indirect targeting such as gene silencing have been explored, these are associated with major drawbacks including weak binding interactions or activity, poor pharmacological properties, and off-target effects. As an alternative approach, the Moellering lab recently developed a new class of hyperstable synthetic DNA-binding domains derived from the transcription factor MAX that orthogonally self- dimerize to sequence-specifically bind DNA with high affinity and specificity. These synthetic transcriptional regulators (STRs) competitively inhibit the DNA binding of native dimers of MYC/MAX or MAX/MAX and contest MYC-dependent gene expression. Building on this precedence, I hypothesize that the potency of these compounds would be increased by covalently ligating split monomeric STRs to produce pre-organized dimeric STRs. Additionally, I hypothesize that generating multi-functional STRs that recruit transcriptional machinery to directed genomic sites would provide further control to inhibit MYC-dependent phenotypes. The proposed research aims to explore these two potential routes for advancing the STR technology and evaluate their effects on MYC-dependent gene expression and phenotypes. Together, this tandem approach will establish a new class of potent MYC antagonists and provide mechanistic tools to study oncogenic gene expression in cells. Additionally, this modular design strategy to develop transcription factor mimetics will provide a framework to enable preparation of future constructs tailored towards other important transcription factors and genomic targets.