Chow Eve W L, Clancey Shelly A, Billmyre R Blake, Averette Anna Floyd, Granek Joshua A, Mieczkowski Piotr, Cardenas Maria E, Heitman Joseph
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.
Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, North Carolina, United States of America.
PLoS Genet. 2017 Apr 4;13(4):e1006667. doi: 10.1371/journal.pgen.1006667. eCollection 2017 Apr.
Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca2+ signaling responses. In Cryptococcus neoformans, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in Saccharomyces cerevisiae and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that loss of Crz1 confers phenotypes intermediate between wild-type and calcineurin mutants, and demonstrate that deletion of the calcineurin docking domain results in the inability of Crz1 to translocate into the nucleus under thermal stress. RNA-sequencing revealed 102 genes that are regulated in a calcineurin-Crz1-dependent manner at 37°C. The majority of genes were down-regulated in cna1Δ and crz1Δ mutants, indicating these genes are normally activated by the calcineurin-Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. We identified three calcineurin-dependent response element motifs within the promoter regions of calcineurin-Crz1 target genes, and show that Crz1 binding to target gene promoters is increased upon thermal stress in a calcineurin-dependent fashion. Additionally, we found a large set of genes independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Given the intermediate crz1Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output in C. neoformans appears to be adapted to promote fungal virulence.
钙调神经磷酸酶是一种高度保守的、依赖Ca2+/钙调蛋白的丝氨酸/苏氨酸特异性蛋白磷酸酶，它协调细胞内Ca2+信号反应。在新型隐球菌中，钙调神经磷酸酶可被包括高温在内的多种应激激活，对压力适应和毒力至关重要。转录因子Crz1是酿酒酵母和其他真菌中的主要钙调神经磷酸酶效应物。钙调神经磷酸酶使Crz1去磷酸化，从而使Crz1发生核转位并转录靶基因。在这里，我们表明Crz1的缺失赋予了介于野生型和钙调神经磷酸酶突变体之间的表型，并证明钙调神经磷酸酶对接结构域的缺失导致Crz1在热应激下无法转运到细胞核中。RNA测序揭示了102个在37°C下以钙调神经磷酸酶-Crz1依赖方式调控的基因。大多数基因在cna1Δ和crz1Δ突变体中下调，表明这些基因通常在高温下被钙调神经磷酸酶-Crz1途径激活。约58%的钙调神经磷酸酶-Crz1靶基因功能未知，而具有已知或预测功能的基因参与细胞壁重塑、钙运输和信息素产生。我们在钙调神经磷酸酶-Crz1靶基因的启动子区域内鉴定出三个钙调神经磷酸酶依赖的反应元件基序，并表明热应激时Crz1以钙调神经磷酸酶依赖的方式增加与靶基因启动子的结合。此外，我们发现了一大组由钙调神经磷酸酶独立调控的基因，并且Crz1独立于钙调神经磷酸酶调控59个基因。鉴于crz1Δ突变体的中间表型，以及我们最近关于钙调神经磷酸酶调控网络独立于Crz1影响P小体和应激颗粒中mRNA的证据，钙调神经磷酸酶可能作用于Crz1之外的因子，这些因子控制mRNA表达/稳定性，以运行真菌毒力所需的分支转录/转录后应激反应网络。综上所述，我们的研究结果表明，从子囊菌到致病担子菌真菌，核心钙调神经磷酸酶-Crz1应激反应级联得以维持，但其在新型隐球菌中的输出似乎经过了适应性调整以促进真菌毒力。
