CRISPR-mediated Chemical Genetics Define Transcription Factor Gene Networks and Mechanisms of Control

摘要 序列特异性转录因子是细胞适应的关键介质 细胞外和细胞内线索。这些快速变化的转录程序促进了一切 细胞命运决定于应激反应，转录因子功能和/或表达的破坏是 与疾病状态相关，包括发育障碍、神经系统疾病和癌症。虽然它是 很明显，基因表达的适当调节对于正常细胞功能至关重要，从历史上看，我们的 能够理解序列特异性转录因子如何快速、特异性地改变转录 程序受到非常缓慢的遗传和敲除策略工具箱的限制，需要几天到几周的时间 在可以测定转录因子活性之前。因此，虽然直接转录效应发生在 从几分钟到几小时，这些模型需要几天的时间才能建立，从而检测到二次和/或 代偿性转录变化通常掩盖转录因子的直接/立即影响 扰乱。为了克服这些技术限制，我们使用 CRISPR 介导的基因组编辑来 将降解决定子标签引入内源转录因子位点。这种化学遗传方法可以快速 PROTAC 处理后转录因子降解（几分钟到几小时），并有效地破坏 检测转录变化、染色质状态和全基因组转录因子的时间范围 占用时间从几天到几小时。我们用基于蛋白质组学的方法来补充这些研究，以识别 相关复合物和协作转录因子，并开始整合单细胞和 单分子成像方法来询问转录反应中的异质性。综合起来， 这些方法使我们能够解决转录领域的基本问题，包括如何 转录因子受染色质景观影响并对染色质景观产生影响，多重序列如何- 特定转录因子协同调节基因表达，以及增强子活性如何影响 启动子激活。

ABSTRACT Sequence-specific transcription factors are critical mediators of cellular adaptation in response to both extracellular and intracellular cues. These rapidly changing transcriptional programs facilitate everything from cell fate decisions to stress responses, and the disruption of transcription factor function and/or expression is associated with disease states including developmental disorders, neurologic disorders, and cancer. While it is clear that the appropriate regulation of gene expression is critical for normal cellular function, historically, our ability to understand how sequence-specific transcription factors rapidly and specifically alter transcriptional programs has been limited by a toolbox of very slow genetic and knockdown strategies that take days to weeks before transcription factor activity can be assayed. Therefore, while direct transcriptional effects occur within minutes to hours, these models take days to establish resulting in the detection of secondary and/or compensatory transcriptional changes that often mask the direct/immediate effects of transcription factor disruption. In order to overcome these technical limitations, we use CRISPR-mediated genome editing to introduce degron tags into endogenous transcription factor loci. This chemical-genetic approach results in rapid transcription factor degradation (minutes to hours) following PROTAC treatment, and effectively collapses the timeframe for assaying transcriptional changes, chromatin states, and genome-wide transcription factor occupancy from days to hours. We complement these studies with proteomics-based approaches to identify associated complexes and cooperating transcription factors, and are beginning to incorporate single cell and single molecule imaging approaches to interrogate heterogeneity within transcriptional responses. Combined, these approaches are allowing us to address fundamental questions in the transcription field including how transcription factors are influenced by and exert influence over the chromatin landscape, how multiple sequence- specific transcription factors cooperate to regulate gene expression, and how enhancer activity influences promoter activation.