Kennedy Patrick H, Deh Sheikh Amin Alborzian, Balakar Matthew, Jones Alexander C, Olive Meagan E, Hegde Mudra, Matias Maria I, Pirete Natan, Burt Rajan, Levy Jonathan, Little Tamia, Hogan Patrick G, Liu David R, Doench John G, Newton Alexandra C, Gottschalk Rachel A, de Boer Carl, Alarcón Suzie, Newby Gregory, Myers Samuel A
bioRxiv. 2023 Nov 14:2023.11.11.566649. doi: 10.1101/2023.11.11.566649.
Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here, we describe "signaling-to-transcription network" mapping through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally-resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of the phosphatase PHLPP1 which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.
驱动基因表达的信号通路通常被描绘为具有大约十几个标志性的磷酸化和转录事件。实际上，数千种动态的翻译后修饰（PTM）几乎调控着每一种细胞功能，而我们缺乏能够大规模地在这些庞大的生化途径和遗传回路之间找到因果联系的技术。在这里，我们描述了通过开发以PTM为中心的碱基编辑并结合表型筛选来绘制“信号转转录网络”，该过程由时间分辨磷酸蛋白质组学指导。以T细胞激活为模型，我们观察到数百个未被研究的磷酸化位点，它们调节NFAT转录活性。我们确定了磷酸酶PHLPP1的磷酸化介导的核定位，其促进NFAT但抑制NFκB活性。我们还发现特定的磷酸位点突变体可以以微妙但独特的模式改变基因表达，这证明了微调转录反应的潜力。总体而言，对PTM位点进行碱基编辑器筛选为剖析信号通路中的PTM功能提供了一个强大的平台。
