In silico design of context-responsive mammalian promoters with user-defined functionality.
In silico design of context-responsive mammalian promoters with user-defined functionality.
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DOI：
10.1093/nar/gkx768
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发表时间：
2017-10-13
影响因子：
14.9
通讯作者：
James DC
中科院分区：
文献类型：
--
作者：
Brown AJ;Gibson SJ;Hatton D;James DC
复杂哺乳动物启动子的全面从头设计受到组成转录因子调控元件（TFRE）之间不可预测的组合相互作用的限制。在这项研究中，我们表明，模块化的结合位点，不合作的功能，可以通过分析宿主细胞转录因子的表达谱，并随后测试同源TFRE活动在不同的同型和异型启动子架构。在特定表达环境中显示位置不敏感的加性功能的TFRE可以在计算机上合理地组合在一起以产生具有高度可预测活性的启动子。由于TFRE顺序和间隔不影响这些TFRE组合的性能，因此可以特别地布置组合物以排除不期望的序列特征的形成。这促进了具有上下文所需的、用户定义的功能的启动子的简单的计算机设计。为了证明这一点，我们重新创建了用于CHO细胞中生物制药生产的启动子，其表现出精确设计的活性动力学和长期表达稳定性，而不会对细胞性能产生可观察到的追溯效应。所描述的设计过程可用于需要上下文响应的、可定制的启动子功能的应用，特别是在合成TF的共表达不合适的情况下。虽然使用的合成启动子结构并不十分类似于原生哺乳动物的架构，我们的研究结果也提供了额外的支持，一个灵活的广告牌模型的启动子调控。
Comprehensive de novo-design of complex mammalian promoters is restricted by unpredictable combinatorial interactions between constituent transcription factor regulatory elements (TFREs). In this study, we show that modular binding sites that do not function cooperatively can be identified by analyzing host cell transcription factor expression profiles, and subsequently testing cognate TFRE activities in varying homotypic and heterotypic promoter architectures. TFREs that displayed position-insensitive, additive function within a specific expression context could be rationally combined together in silico to create promoters with highly predictable activities. As TFRE order and spacing did not affect the performance of these TFRE-combinations, compositions could be specifically arranged to preclude the formation of undesirable sequence features. This facilitated simple in silico-design of promoters with context-required, user-defined functionalities. To demonstrate this, we de novo-created promoters for biopharmaceutical production in CHO cells that exhibited precisely designed activity dynamics and long-term expression-stability, without causing observable retroactive effects on cellular performance. The design process described can be utilized for applications requiring context-responsive, customizable promoter function, particularly where co-expression of synthetic TFs is not suitable. Although the synthetic promoter structure utilized does not closely resemble native mammalian architectures, our findings also provide additional support for a flexible billboard model of promoter regulation.
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影响因子：
64.8
作者：
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DOI：
10.1016/j.csbj.2014.07.005
发表时间：
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