Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA.
Department of Bioengineering, University of California San Diego, La Jolla, CA 92093, USA.
Nucleic Acids Res. 2018 Nov 16;46(20):10682-10696. doi: 10.1093/nar/gky752.
Transcriptional regulation enables cells to respond to environmental changes. Of the estimated 304 candidate transcription factors (TFs) in Escherichia coli K-12 MG1655, 185 have been experimentally identified, but ChIP methods have been used to fully characterize only a few dozen. Identifying these remaining TFs is key to improving our knowledge of the E. coli transcriptional regulatory network (TRN). Here, we developed an integrated workflow for the computational prediction and comprehensive experimental validation of TFs using a suite of genome-wide experiments. We applied this workflow to (i) identify 16 candidate TFs from over a hundred uncharacterized genes; (ii) capture a total of 255 DNA binding peaks for ten candidate TFs resulting in six high-confidence binding motifs; (iii) reconstruct the regulons of these ten TFs by determining gene expression changes upon deletion of each TF and (iv) identify the regulatory roles of three TFs (YiaJ, YdcI, and YeiE) as regulators of l-ascorbate utilization, proton transfer and acetate metabolism, and iron homeostasis under iron-limited conditions, respectively. Together, these results demonstrate how this workflow can be used to discover, characterize, and elucidate regulatory functions of uncharacterized TFs in parallel.
转录调控使细胞能够对外界环境变化做出响应。在大肠杆菌 K-12 MG1655 中估计有 304 个候选转录因子 (TFs)，其中 185 个已经通过实验鉴定，但 ChIP 方法仅对少数几十个进行了全面表征。鉴定这些剩余的 TF 是提高我们对大肠杆菌转录调控网络 (TRN) 认识的关键。在这里，我们开发了一种综合工作流程，用于使用一系列全基因组实验对 TF 进行计算预测和全面的实验验证。我们将此工作流程应用于：(i) 从一百多个未表征的基因中鉴定出 16 个候选 TF；(ii) 总共捕获了十个候选 TF 的 255 个 DNA 结合峰，得出了六个高可信度的结合基序；(iii) 通过确定每个 TF 缺失时基因表达的变化，重建这十个 TF 的调控网络；(iv) 确定三个 TF (YiaJ、YdcI 和 YeiE) 的调节作用，分别为 l-抗坏血酸利用、质子转移和铁限制条件下的乙酸盐代谢以及铁稳态的调节剂。总之，这些结果表明了这种工作流程如何能够用于平行发现、表征和阐明未表征 TF 的调控功能。
