Identification and characterization of sugar regulated promoters in Chaetomium thermophilum

The thermophilic fungus Chaetomium thermophilum has been successfully used in the past for biochemical and high resolution structural studies of protein complexes, but subsequent functional analysis of these assemblies were hindered due to the lack of genetic tools in this thermophile, which are typically amenable in several other mesophilic eukaryotic model organisms, in particular the yeast Saccharomycers cerevisiae. Hence, we aimed to develop a regulatable gene-expression system in C. thermophilum, which might facilitate such in vivo studies, based on what we know about the galactose-inducible GAL promoter in yeast. To identify sugar-regulatable promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes induced by xylose but repressed by glucose. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of the thermostable YFP (yellow fluorescent protein) reporter. In this way, we could demonstrate xylose-dependent YFP expression by either western blotting or life cell imaging fluorescence microscopy. Prompted by these results, we finally expressed a well-characterized dominant-negative ribosome assembly factor mutant, rsa4 E117>D, under the control of the XDH promoter, which allowed us to induce a nuclear export defect of the pre-60S subunit when C. thermophilum cells were grown in xylose but not glucose containing medium. Altogether, our study recognized xylose-regulatable promoters in Chaetomium thermophilum, which may foster functional studies of genes of interest in this thermophilic eukaryotic model organism. Overall design: Comparative gene expression profiling analysis of RNA-seq data for Chaetomium thermophilum cells exposed to glucose (G) or xylose (X) as sole carbon source in comparison to a carbon depleted refernece medium (R).

嗜热真菌嗜热毛壳菌（Chaetomium thermophilum）此前已成功应用于蛋白质复合物的生化分析与高分辨率结构研究，但由于该嗜热菌缺乏遗传工具——这类工具在诸多其他中温真核模式生物中通常可便捷获取，尤其是酿酒酵母（Saccharomyces cerevisiae）——这类复合物组装体的后续功能分析受到了阻碍。因此，基于我们对酿酒酵母半乳糖诱导型GAL启动子的研究基础，我们旨在开发嗜热毛壳菌中的可调控基因表达系统，以推动此类体内功能研究。为鉴定嗜热毛壳菌中的糖调控型启动子，我们开展了木糖与葡萄糖依赖性基因表达的对比分析，筛选出一批受木糖诱导、葡萄糖抑制的酶类。随后，我们将全基因组分析中获得的两个调控最为严格的基因——木糖苷酶样基因（XYL）与木糖醇脱氢酶（XDH）——的启动子，克隆至耐热黄色荧光蛋白（yellow fluorescent protein, YFP）报告基因的上游区域。通过该系统，我们可通过蛋白质免疫印迹（western blotting）或活细胞成像荧光显微镜，检测到木糖依赖的YFP表达。受上述结果启发，我们最终在XDH启动子的调控下，表达了一个特征明确的显性负突变核糖体组装因子rsa4 E117>D。当嗜热毛壳菌在含木糖而非葡萄糖的培养基中培养时，该系统可诱导前60S核糖体亚基出现核输出缺陷。综上，本研究成功鉴定出嗜热毛壳菌中的木糖调控型启动子，这将有力推动该嗜热真核模式生物中目标基因的功能研究。整体实验设计：以碳源匮乏参照培养基（R）为对照，对以葡萄糖（G）或木糖（X）作为唯一碳源培养的嗜热毛壳菌细胞的RNA测序数据，开展比较基因表达谱分析。