The Compendium of DNA-Binding Specificities of Transcription Factors in a Pathogenic Bacterium

The regulation of transcription is primarily exerted through transcription factors (TFs) binding to genomic DNA. Although molecular mechanisms of TFs have been studied individually for decades, a complete picture of binding profiles of all TFs and their precise targets in the genome are still lacking in the model pathogen Pseudomonas syringae. To this end, we performed a high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX) approach on all 301 annotated TFs in P. syringae. Robust enrichment of specific sequences was deduced to 118 SELEX motifs. We identified 12,464 interactions between 100 TFs and their target genes in the genome, for an individual TF ranging from 6 to 1481 sites. It showed that 90% TF binding was of dimeric site type, in which 85% with the head-to-head palindromic binding preference. To further explore the pathogenic mechanism of TFs in P. syringae, we mapped intricate networks of these TFs and their targets in the virulence-associated pathways, many of which were verified by orthologous methods such as ChIP-seq, EMSA, RT-qPCR and a reporter assay of promoter activity. By checking the enrichment of binding sites in pathways, we identified 25 virulence-associated master regulators of which 14 had never been characterized as TFs before. Overall, the present study provides a valuable resource for TF binding specificities in P. syringae and demonstrates a novel and an integrative analysis for seeking the virulence-associated TFs and its target genes. We expect that the results will significantly benefit future studies on the transcriptional regulation in P. syringae, and facilitate the design of drug targets to protect plants from attacks by relevant pathogens. Overall design: In order to globally profile the DNA binding specificities of all TFs in P. syringae, we purified the proteins for all 301 annotated TFs in the genome and applied a high-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX) approach.

转录调控主要通过转录因子（transcription factors, TFs）结合基因组DNA来实现。尽管转录因子的分子机制已被单独研究数十年，但模式病原菌丁香假单胞菌（Pseudomonas syringae）中，所有转录因子的结合谱及其在基因组中的精准靶标仍未得到完整阐明。为此，我们针对丁香假单胞菌基因组中注释的全部301个转录因子，开展了高通量指数富集配体系统进化（high-throughput systematic evolution of ligands by exponential enrichment, HT-SELEX）实验。我们将特异性序列的有效富集结果归纳为118个SELEX基序。我们鉴定出100个转录因子与其基因组靶基因之间的12464个互作关系，单个转录因子的结合位点数量介于6至1481个之间。研究显示，90%的转录因子结合位点为二聚体类型，其中85%具有头对头回文结构的结合偏好。为进一步探究丁香假单胞菌中转录因子的致病机制，我们绘制了这些转录因子及其靶标在毒力相关通路中的复杂调控网络，其中多数关联已通过染色质免疫沉淀测序（ChIP-seq）、电泳迁移率变动分析（EMSA）、实时定量PCR（RT-qPCR）以及启动子活性报告基因实验等同源验证方法得到验证。通过检测结合位点在通路中的富集情况，我们鉴定出25个毒力相关的主控调控因子，其中14个此前从未被鉴定为转录因子。总体而言，本研究为丁香假单胞菌的转录因子结合特异性提供了宝贵的研究资源，并为挖掘毒力相关转录因子及其靶基因提供了一种全新的整合分析策略。我们预期该研究结果将显著推动后续丁香假单胞菌转录调控相关研究，并助力设计靶向药物以保护植物免受相关病原菌侵染。总体实验设计：为全面解析丁香假单胞菌所有转录因子的DNA结合特异性，我们纯化了该基因组中注释的全部301个转录因子蛋白，并应用了高通量指数富集配体系统进化（HT-SELEX）技术。