SINGLE-CELL CHEMICAL TRANSCRIPTOMIC DISSECTION OF AN ESSENTIAL TRANSCRIPTION FACTOR NETWORK

项目摘要/摘要 碱性螺旋-环-螺旋亮氨酸拉链转录因子(TF)在延伸的MYC网络中起着至关重要的作用 调节细胞生长、分化和动态平衡的作用。开创性研究表明MYC 及其相互作用的转录因子网络，作为发育和癌症中增殖和新陈代谢的驱动力，但 这些因子的剂量如何编码转录状态仍然存在争议。在这里，我提议写一部小说 化学基因组框架，系统地确定转铁蛋白的剂量、转录产量和 蜂窝状态是相关联的。我将结合我们最近发表的大规模多路单细胞rna-seq 化学遗传降解蛋白质靶标的筛选方法(SCI-Plex) 在TF的剂量降解之后，胚胎干细胞在转录上被重新编程。那我会的 使用最先进的计算工具来量化特定编码的蜂窝状态轨迹 剂量。最后，我将利用这个单细胞‘微扰图谱’来支持基因组图谱实验 为了了解这些因子在体内的剂量减少如何导致物理上的再分布 基因组。这些研究将用于。)阐明Tf剂量编码转录输出的机制， II.)阐明相互作用的转录因子的细胞内浓度如何维持染色质、转录和 细胞状态和ii.)提供转录因子网络如何与转录因子相互作用的机械性见解 表观基因组来调节哺乳动物的多能性。更广泛地说，这些方法有可能 解决长期存在的基因调控问题，即蛋白质剂量如何控制两者的细胞状态 健康和癌症。

PROJECT SUMMARY / ABSTRACT Basic helix-loop-helix leucine zipper transcription factors (TFs) in the extended MYC network play essential roles regulating cellular growth, differentiation, and homeostasis. Seminal studies have implicated MYC and its interacting network of TFs as drivers of proliferation and metabolism in development and cancer, but how dosage of these factors encodes transcriptional state remains contentious. Here, I propose a novel chemical genomic framework to systematically determine how TF dosage, transcriptional output, and cellular state are linked. I will combine our recently published massively multiplex single-cell RNA-seq screening method (sci-Plex) with chemical genetic degradation of protein targets to examine how embryonic stem cells are transcriptionally reprogrammed following dosed degradation of TFs. I will then employ state-of-the-art computational tools to quantify the cellular state trajectories encoded by specific dosages. Finally, I will leverage this single-cell ‘perturbation atlas’ to support genomic mapping experiments to understand how reduced dosage of these factors in vivo leads to physical redistribution across the genome. These studies will i.) illuminate mechanisms by which TF dosages encode transcriptional output, ii.) elucidate how intracellular concentrations of interacting TFs maintain chromatin, transcriptional, and cellular states, and iii.) provide mechanistic insight into how a transcription factor network interacts with the epigenome to regulate mammalian pluripotency. More generally, these approaches have the potential to address long-standing gene regulatory questions of how protein dosage controls cellular state in both health and in cancer.