Identification and characterization of sugar regulated promoters in Chaetomium thermophilum. 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（尤其是酿酒酵母Saccharomyces cerevisiae）中已成熟可用——导致后续针对这些复合物的功能分析受阻。
因此，本研究基于对酿酒酵母半乳糖诱导型GAL启动子（galactose-inducible GAL promoter）的认知，旨在开发一套适用于嗜热毛壳菌的可调控基因表达系统，以推动相关体内研究的开展。
为在嗜热毛壳菌中鉴定糖调控型启动子，我们开展了木糖与葡萄糖依赖型基因表达的比较分析，筛选出一批受木糖诱导、葡萄糖抑制的酶类。
随后，我们将基因组范围分析中筛选出的两个调控最严格的基因——木糖苷酶样基因（xylosidase-like gene, XYL）与木糖醇脱氢酶（xylitol dehydrogenase, XDH）——的启动子，克隆至热稳定黄色荧光蛋白（yellow fluorescent protein, YFP）报告基因的上游区域。
通过该系统，我们可借助蛋白质印迹（western blotting）或活细胞成像荧光显微镜术，观测到木糖依赖型的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效核糖体组装因子突变体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，该突变体可诱导核糖体60S前亚基的核输出缺陷。
综上，本研究在嗜热毛壳菌中鉴定出了木糖调控型启动子，这将助力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：以碳缺失参照培养基为对照，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进行RNA测序（RNA-seq）数据的比较基因表达谱分析。