Mediator复合物在里氏木霉纤维素酶基因转录表达中的作用调控机制

Co-activators play a central role in transcriptional activation and regulation of eukaryotic genes. The Mediator complex is a functionally conserved co-activator complex that exists extensively in eukaryotic organisms. Our previous study found that deletion of the Mediator tail-module subunit Gal11 resulted in a dramatically decreased expression of cellulase genes in filamentous fungi Trichoderma reesei. Unexpectedly, further analysis revealed that the occupation of the key transcriptional activator Xyr1 on cellulse gene promoter was much higher in the Δgal11 mutant than in the wild type strain, indicating that the Mediator complex play a critical role in the induced expression of cellualse genes in T. reesei, and there may exist a delicate interrelationship between Mediator and Xyr1. This application aims to elucidate the mechanism underlying the action of the Mediator complex in cellulase gene regulation. Systematic studies will be firstly carried out to analyze the genetic function of this complex upon cellulose induction. Further investigation will be conducted to uncover the mechanism by which the Mediator acts on transcriptional activation of cellulase genes and the potential feed-back regulation of Xyr1 activity. The results are anticipated not only to extend the understanding of the Mediator role in transcriptional gene activation, but also to provide deeper insights into the regulation system for T. reesei cellulase synthesis, and to help genetically engineer T. reesei strains to improve cellulase production.

Mediator复合物是广泛存在于真核生物的一个功能保守复合物，在基因转录起始过程中发挥传递转录激活信号的桥梁作用。我们在前期研究发现，在代表性丝状真菌里氏木霉中，Mediator复合物Gal11亚基的缺失导致核心纤维酶基因转录水平特异性下调，而转录激活因子Xyr1在纤维素酶基因启动子的募集水平却显著提高。本项目拟围绕该复合物在里氏木霉纤维素酶基因转录激活中的功能和作用机制展开研究，首先明确Mediator复合物在纤维素酶基因诱导表达过程中的功能，并解析该复合物与Xyr1之间的相互作用关系以及在纤维素酶基因启动子上的招募关系，在此基础上阐明复合物对Xyr1活性的调控作用及作用机制。该研究将极大地促进对丝状真菌纤维素酶基因诱导表达调控机制的认识，并为构建纤维素酶高产菌株提供理论依据和指导。

木质纤维素作为地球上含量最为丰富的生物质资源，其有效利用对于维持社会经济可持续发展和可再生清洁能源的开发具有重大战略意义。丝状真菌里氏木霉是自然界最高效的木质纤维素降解真菌之一，并且作为纤维素酶高效生产菌株目前已广泛应用于生物质能源开发等工业领域。但是目前为止对于其纤维素酶基因高效诱导表达的调控机制认不清楚。在模式真菌酿酒酵母中，Mediator复合物广泛参与了受环境条件诱导表达基因的转录激活调控，但在里氏木霉中其生理学功能及其是否直接参与纤维素酶系基因的高效转录激活尚未见报道。本项目研究发现，在里氏木霉中，Mediator复合物的一个负责介导与转录因子互作的亚基Gal11的缺失会导致内切和外切纤维酶基因的诱导转录水平急剧下调，但并不影响β-葡萄糖苷酶基因的表达，表明Gal11特异性地只参与核心纤维素酶基因的诱导激活。Gal11缺失造成的核心纤维素酶基因的诱导表达缺陷无法通过过表达关键转录激活因子Xyr1来恢复，暗示Gal11在Xyr1介导的转录激活过程中发挥了关键作用。生化分析发现，Gal11和Xyr1之间存在直接相互作用，并且体内染色质免疫共沉淀分析发现，Gal11能以Xyr1依赖的方式被招募至纤维素酶基因的启动子区直接参与其转录激活。进一步分析发现，Gal11在核心纤维素酶基因启动子区RNA聚合酶II的募集中发挥了关键作用，表明Xyr1在介导转录激活过程中很大程度上是通过招募Gal11-Mediator复合物来实现的。对Mediator复合物其他模块亚基的功能进行分析发现，激酶模块亚基Srb10和尾部模块亚基Med5在纤维素酶基因转录激活中也发挥了重要调控作用，这表明Mediator复合物可能作为一个整体参与了里氏木霉纤维素酶基因的高效诱导表达。上述研究工作加深了里氏木霉纤维素酶基因的诱导表达调控机制的认识，为里氏木霉菌株的遗传改良提供了理论指导。