Design of Large-Scale Reporter Construct Arrays for Dynamic, Live Cell Systems Biology.
Design of Large-Scale Reporter Construct Arrays for Dynamic, Live Cell Systems Biology.
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用于动态活细胞系统生物学的大规模报告基因构建阵列的设计。
DOI：
10.1021/acssynbio.8b00236
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发表时间：
2018
影响因子：
4.7
通讯作者：
Shea,LonnieD
中科院分区：
文献类型：
--
作者：
Decker,JosephT;Hall,MatthewS;Peñalver-Bernabé,Beatriz;Blaisdell,RachelB;Liebman,LaurenN;Jeruss,JacquelineS;Shea,LonnieD
动态系统生物学旨在通过对活细胞的分析来确定决定细胞命运的分子机制。在CRISPR/Cas9直接插入荧光素酶之后，可以从提供单个转录因子或与完整启动子结合的多个因子的活性的报告构建物中获得来自活细胞的大规模分子信息。在这份报告中，我们调查了设计标准，以获得对转录因子(TF)结合以及转录因子结合活性和遗传报告活性的整合具有特异性和响应性的报告。利用具有随机组成和长度的间隔物的25个Smad3活性报告基因文库，研究了一致结合位点间隔序列和非靶点结合对报告灵敏度的影响。需要一个间隔区来量化转化生长因子β刺激后的活性变化。TF结合位点预测算法(BEEML、FIMO和DeepBind)用于预测脱靶结合，对Smad3报告的无反应与预测的结构性活性p53的竞争结合相关。Smad3报告基因的活性网络是从Tf报告文库的测量中推断出来的，并与大规模的遗传报告基因活性测量相联系。转录因子和遗传记者的整合确定了指导对转化生长因子β反应的主要枢纽，该方法提供了一个系统水平的算法来研究细胞信号转导。
Dynamic systems biology aims to identify the molecular mechanisms governing cell fate decisions through the analysis of living cells. Large scale molecular information from living cells can be obtained from reporter constructs that provide activities for either individual transcription factors or multiple factors binding to the full promoter following CRISPR/Cas9 directed insertion of luciferase. In this report, we investigated the design criteria to obtain reporters that are specific and responsive to transcription factor (TF) binding and the integration of TF binding activity with genetic reporter activity. The design of TF reporters was investigated for the impact of consensus binding site spacing sequence and off-target binding on the reporter sensitivity using a library of 25 SMAD3 activity reporters with spacers of random composition and length. A spacer was necessary to quantify activity changes after TGFβ stimulation. TF binding site prediction algorithms (BEEML, FIMO and DeepBind) were used to predict off-target binding, and nonresponsiveness to a SMAD3 reporter was correlated with a predicted competitive binding of constitutively active p53. The network of activity of the SMAD3 reporter was inferred from measurements of TF reporter library, and connected with large-scale genetic reporter activity measurements. The integration of TF and genetic reporters identified the major hubs directing responses to TGFβ, and this method provided a systems-level algorithm to investigate cell signaling.