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从 细胞命运决定应激反应，以及转录因子功能和/或表达的破坏， 与包括发育障碍、神经障碍和癌症的疾病状态相关。虽然 很明显，基因表达的适当调节对正常细胞功能至关重要，从历史上看，我们 理解序列特异性转录因子如何快速特异性地改变转录水平的能力 程序一直受到非常缓慢的遗传和击倒策略工具箱的限制，这些策略需要几天到几周的时间 然后才能测定转录因子的活性。因此，虽然直接的转录效应发生在 几分钟到几小时，这些模型需要几天的时间才能建立，从而检测到次级和/或 通常掩盖转录因子的直接/即时效应的补偿性转录变化 破坏为了克服这些技术限制，我们使用CRISPR介导的基因组编辑， 将降解决定子标签引入内源转录因子基因座。这种化学遗传方法导致快速的 在PROTAC处理后的转录因子降解（分钟至小时），并有效地瓦解了 检测转录变化、染色质状态和全基因组转录因子的时间框架 从几天到几小时的占用。我们补充这些研究与蛋白质组学为基础的方法来确定 相关的复合物和合作转录因子，并开始纳入单细胞和 单分子成像方法来询问转录应答内的异质性。结合起来， 这些方法使我们能够解决转录领域的基本问题， 转录因子受染色质景观的影响，并对染色质景观施加影响，多个序列- 特异性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.