Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada.
PLoS Pathog. 2011 Sep;7(9):e1002266. doi: 10.1371/journal.ppat.1002266. Epub 2011 Sep 29.
In F. graminearum, the transcriptional regulator Tri6 is encoded within the trichothecene gene cluster and regulates genes involved in the biosynthesis of the secondary metabolite deoxynivalenol (DON). The Tri6 protein with its Cys₂His₂ zinc-finger may also conform to the class of global transcription regulators. This class of global transcriptional regulators mediate various environmental cues and generally responds to the demands of cellular metabolism. To address this issue directly, we sought to find gene targets of Tri6 in F. graminearum grown in optimal nutrient conditions. Chromatin immunoprecipitation followed by Illumina sequencing (ChIP-Seq) revealed that in addition to identifying six genes within the trichothecene gene cluster, Tri1, Tri3, Tri6, Tri7, Tri12 and Tri14, the ChIP-Seq also identified 192 additional targets potentially regulated by Tri6. Functional classification revealed that, among the annotated genes, ∼40% are associated with cellular metabolism and transport and the rest of the target genes fall into the category of signal transduction and gene expression regulation. ChIP-Seq data also revealed Tri6 has the highest affinity toward its own promoter, suggesting that this gene could be subject to self-regulation. Electro mobility shift assays (EMSA) performed on the promoter of Tri6 with purified Tri6 protein identified a minimum binding motif of GTGA repeats as a consensus sequence. Finally, expression profiling of F. graminearum grown under nitrogen-limiting conditions revealed that 49 out of 198 target genes are differentially regulated by Tri6. The identification of potential new targets together with deciphering novel binding sites for Tri6, casts new light into the role of this transcriptional regulator in the overall growth and development of F. graminearum.
在禾谷镰刀菌中,转录调控因子 Tri6 编码在三萜烯基因簇内,并调节次级代谢产物脱氧雪腐镰刀菌烯醇 (DON) 生物合成相关基因。Tri6 蛋白及其 Cys₂His₂锌指结构可能也符合全局转录调控因子的类别。这类全局转录调控因子介导各种环境线索,通常响应细胞代谢的需求。为了直接解决这个问题,我们试图在禾谷镰刀菌最佳营养条件下生长时找到 Tri6 的基因靶点。染色质免疫沉淀 followed by Illumina 测序(ChIP-Seq)表明,除了在三萜烯基因簇内鉴定出六个基因(Tri1、Tri3、Tri6、Tri7、Tri12 和 Tri14)外,ChIP-Seq 还鉴定出 192 个潜在受 Tri6 调控的额外靶点。功能分类表明,在注释基因中,约 40%与细胞代谢和运输有关,其余靶基因属于信号转导和基因表达调控类别。ChIP-Seq 数据还表明 Tri6 对其自身启动子具有最高亲和力,表明该基因可能受到自身调控。用纯化的 Tri6 蛋白在 Tri6 启动子上进行的电泳迁移率变动分析(EMSA)确定了 GTGA 重复作为一个共识序列的最小结合基序。最后,在氮限制条件下生长的禾谷镰刀菌的表达谱分析表明,198 个靶基因中有 49 个受 Tri6 差异调控。潜在新靶点的鉴定以及 Tri6 新结合位点的破译,为该转录调控因子在禾谷镰刀菌整体生长和发育中的作用提供了新的认识。