Optogenetic control of RelA reveals effect of transcription factor dynamics on downstream gene expression
Optogenetic control of RelA reveals effect of transcription factor dynamics on downstream gene expression
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DOI：
10.1101/2022.08.03.502739
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发表时间：
2022-08
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影响因子：
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通讯作者：
Lindsey C. Osimiri;Alain R. Bonny;S. Takagishi;Stefanie Luecke;Nina Riehs;Alexander Hoffmann;H. El-Samad
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作者：
Lindsey C. Osimiri;Alain R. Bonny;S. Takagishi;Stefanie Luecke;Nina Riehs;Alexander Hoffmann;H. El-Samad
许多转录因子（TF）响应不同的输入以不同的动态模式易位到细胞核。这种行为的一个显着例子是 RelA，它是 NF-κB 的一个亚基，它根据刺激以脉冲或持续的动态方式易位到细胞核。我们对下游基因如何解释这些动态的理解仍然不完整，部分原因是普遍使用的环境输入激活了除 RelA 之外的其他转录调节因子。在这里，我们使用光遗传学工具 CLASP（可控光激活穿梭和质膜隔离）来控制小鼠成纤维细胞中的 RelA 时空动力学，并使用 RNA-seq 量化其对下游基因的影响。使用 RelA-CLASP，我们首次证明 RelA 的核转位，无需翻译后修饰或激活其他转录调节因子，足以激活下游基因。此外，我们发现TNFα作为一种常见的内源性输入，独立于RelA调节许多基因，并且这种基因调节与RelA-CLASP诱导的基因调节不同。对 RelA-CLASP 响应的基因在响应恒定的 RelA 输入时表现出广泛的动态变化。我们使用简单的启动子模型来概括这些不同的动态响应，以及响应脉冲 RelA-CLASP 输入而收集的数据，并提取许多 RelA 响应启动子的特征。我们还查明了许多基因，可能需要更复杂的模型（涉及反馈或多步启动子）来解释它们对恒定和脉冲 TF 输入的反应。这项研究引入了一种用于研究哺乳动物转录调控的新的强大工具，并展示了光遗传学工具在剖析重要细胞途径的定量特征方面的力量。
Many transcription factors (TFs) translocate to the nucleus with varied dynamic patterns in response to different inputs. A notable example of such behavior is RelA, a subunit of NF-κB, which translocates to the nucleus with either pulsed or sustained dynamics, depending on the stimulus. Our understanding of how these dynamics are interpreted by downstream genes has remained incomplete, partly because ubiquitously used environmental inputs activate other transcriptional regulators in addition to RelA. Here, we use an optogenetic tool, CLASP (controllable light-activated shuttling and plasma membrane sequestration), to control RelA spatiotemporal dynamics in mouse fibroblasts and quantify their effect on downstream genes using RNA-seq. Using RelA-CLASP, we show for the first time that nuclear translocation of RelA, without post-translational modifications or activation of other transcriptional regulators, is sufficient to activate downstream genes. Furthermore, we find that TNFα, a common endogenous input, regulates many genes independently of RelA, and that this gene regulation is different from that induced by RelA-CLASP. Genes responsive to RelA-CLASP show a wide range of dynamics in response to a constant RelA input. We use a simple promoter model to recapitulate these diverse dynamic responses, as well as data collected in response to a pulsed RelA-CLASP input, and extract features of many RelA-responsive promoters. We also pinpoint many genes for which more complex models, involving feedback or multi-step promoters, may be needed to explain their response to constant and pulsed TF inputs. This study introduces a new robust tool for studying mammalian transcriptional regulation and demonstrates the power of optogenetic tools in dissecting the quantitative features of important cellular pathways.