Evolution of Reduced Co-Activator Dependence Led to Target Expansion of a Starvation Response Pathway [Cgla RNA-seq]

In S. cerevisiae, the phosphate starvation (PHO) responsive transcription factors Pho4 and Pho2 are jointly required for induction of phosphate response genes and survival in phosphate starvation conditions. In the related human commensal and pathogen C. glabrata, Pho4 is required but Pho2 is dispensable for survival in phosphate-limited conditions and is only partially required for inducing the phosphate response genes. This reduced dependence on Pho2 evolved in C. glabrata and closely related species. Pho4 orthologs that are less dependent on Pho2 induce more genes when introduced into the S. cerevisiae background, and Pho4 in C. glabrata both binds to more sites and induces more genes with expanded functional roles compared to Pho4 in S. cerevisiae. We used RNA-seq to profile the transcriptome of wild type and mutants of Pho4 / Pho2 in C. glabrata, to identify genes induced by Pho4. Overall design: The goal is to identify genes induced by Pho4 in C. glabrata (CgPho4), either with or without C. glabrata Pho2 (CgPho2) in a genetic background where the negative regulator of Pho4, Pho80, is deleted. Pairwise comparisons such as that between PHO4 wild-type and pho4? strain, in the pho80? CgPHO2 background, will identify genes induced by Pho4 in the presence of Pho2. For each strain, two biological replicates, i.e. the same genotype grown, collected and processed separately, were analyzed by RNA-seq.

在酿酒酵母（S. cerevisiae）中，磷饥饿（PHO）响应转录因子Pho4与Pho2共同介导磷饥饿响应基因的诱导，并在磷饥饿条件下保障细胞存活。在亲缘关系相近的人类共生兼致病菌光滑念珠菌（C. glabrata）中，Pho4是维持磷限制条件下细胞存活所必需的，而Pho2则并非必需，且仅部分参与磷饥饿响应基因的诱导过程。这种对Pho2依赖性的降低，是在光滑念珠菌及其近缘物种中演化形成的。相较于酿酒酵母中的Pho4，对Pho2依赖性更低的Pho4同源蛋白，在被引入酿酒酵母遗传背景后可诱导更多基因的表达；而光滑念珠菌中的Pho4不仅结合的靶位点更多，还可诱导更多功能范围得到扩展的基因。本研究通过RNA测序（RNA-seq）对光滑念珠菌中Pho4/Pho2的野生型与突变体的转录组进行分析，以鉴定受Pho4诱导的基因。总体实验设计：本研究旨在鉴定光滑念珠菌中Pho4（CgPho4）所诱导的基因，实验分别在存在或缺失光滑念珠菌Pho2（CgPho2）的遗传背景下开展，且所有实验均在Pho4的负调控因子Pho80被敲除的遗传背景中进行。在Pho80敲除（pho80Δ）且保留功能性CgPHO2的背景下，通过野生型PHO4菌株与pho4Δ缺失突变株的两两比较，可鉴定出Pho2存在时由Pho4诱导的基因。针对每一种菌株，均设置两份生物学重复，即相同基因型的菌体分别独立培养、收集并完成样本处理，随后通过RNA-seq完成转录组分析。