BACKGROUND The calcium/calcineurin signaling pathway is mediated by the transcription factors NFAT (nuclear factor of activated T cells) in mammals and Crz1 (calcineurin-responsive zinc finger 1) in yeasts and other lower eukaryotes. A previous microarray analysis identified a putative Crz1-binding motif in promoters of its target genes in Candida albicans, but it has not been experimentally demonstrated. METHODS An inactivation mutant for CaCRZ1 was generated through CRISPR/Cas9 approach. Transcript profiling was carried out by RNA sequencing of the wild type and the inactivation mutant for CaCRZ1 in response to 0.2 M CaCl2. Gene promoters were scanned by the online MEME (Multiple Em for Motif Elicitation) software. Gel electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were used for in vitro and in vivo CaCrz1-binding experiments, respectively. RESULTS RNA sequencing reveals that expression of 219 genes is positively, and expression of 59 genes is negatively, controlled by CaCrz1 in response to calcium stress. These genes function in metabolism, cell cycling, protein fate, cellular transport, signal transduction, transcription, and cell wall biogenesis. Forty of these positively regulated 219 genes have previously been identified by DNA microarray analysis. Promoter analysis of these common 40 genes reveals a consensus motif [5'-GGAGGC(G/A)C(T/A)G-3'], which is different from the putative CaCrz1-binding motif [5'-G(C/T)GGT-3'] identified in the previous study, but similar to Saccharomyces cerevisiae ScCrz1-binding motif [5'-GNGGC(G/T)CA-3']. EMSA and ChIP assays indicate that CaCrz1 binds in vitro and in vivo to both motifs in the promoter of its target gene CaUTR2. Promoter mutagenesis demonstrates that these two CaCrz1-binding motifs play additive roles in the regulation of CaUTR2 expression. In addition, the CaCRZ1 gene is positively regulated by CaCrz1. CaCrz1 can bind in vitro and in vivo to its own promoter, suggesting an autoregulatory mechanism for CaCRZ1 expression. CONCLUSIONS CaCrz1 differentially binds to promoters of its target genes to regulate their expression in response to calcium stress. CaCrz1 also regulates its own expression through the 5'-TGAGGGACTG-3' site in its promoter. Video abstract.

中文翻译：

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RNA测序揭示了人类真菌病原体白色念珠菌靶基因启动子中的一个Crz1结合基序。

背景技术钙/钙调神经磷酸酶的信号传导途径是由哺乳动物的转录因子NFAT（活化的T细胞的核因子）和酵母和其他低等真核生物的Crz1（钙调神经磷酸酶的锌指1）介导的。先前的微阵列分析在白色念珠菌的靶基因启动子中鉴定出推定的Crz1结合基序，但尚未通过实验证明。方法通过CRISPR / Cas9方法产生CaCRZ1的失活突变体。通过野生型和响应0.2 M CaCl2的CaCRZ1失活突变体的RNA测序，进行转录谱分析。基因启动子通过在线MEME（用于母体激发的Multi Em）软件进行扫描。凝胶电泳迁移率变动分析（EMSA）和染色质免疫沉淀（ChIP）分析分别用于体外和体内CaCrz1结合实验。结果RNA测序显示，CaCaz1响应钙胁迫而控制了219个基因的阳性表达，而59个基因的阴性表达。这些基因在新陈代谢，细胞周期，蛋白质命运，细胞转运，信号转导，转录和细胞壁生物发生中起作用。这些微调的219个基因中有40个先前已通过DNA微阵列分析鉴定。对这些常见的40个基因的启动子分析揭示了一个共有基序[5'-GGAGGC（G / A）C（T / A）G-3']，与假定的CaCrz1结合基序[5'-G（C / T）GGT-3']，但类似于酿酒酵母ScCrz1结合基序[5'-GNGGC（G / T）CA-3']。EMSA和ChIP分析表明，CaCrz1在体外和体内均与目标基因CaUTR2启动子中的两个基序结合。启动子诱变表明，这两个CaCrz1绑定基序在CaUTR2表达的调节中起附加作用。此外，CaCRZ1基因受到CaCrz1的正调控。CaCrz1可以在体外和体内与其自身的启动子结合，提示CaCRZ1表达的自调节机制。结论CaCrz1差异性结合其靶基因的启动子，以响应钙胁迫调节其表达。CaCrz1还通过其启动子中的5'-TGAGGGACTG-3'位调节其自身表达。录像摘要。EMSA和ChIP分析表明，CaCrz1在体外和体内与目标基因CaUTR2启动子中的两个基序结合。启动子诱变表明，这两个CaCrz1绑定基序在CaUTR2表达的调节中起附加作用。此外，CaCRZ1基因受到CaCrz1的正调控。CaCrz1可以在体外和体内与其自身的启动子结合，提示CaCRZ1表达的自调节机制。结论CaCrz1差异性结合其靶基因的启动子，以响应钙胁迫调节其表达。CaCrz1还通过其启动子中的5'-TGAGGGACTG-3'位调节其自身表达。录像摘要。EMSA和ChIP分析表明，CaCrz1在体外和体内与目标基因CaUTR2启动子中的两个基序结合。启动子诱变表明，这两个CaCrz1绑定基序在CaUTR2表达的调节中起附加作用。此外，CaCRZ1基因受到CaCrz1的正调控。CaCrz1可以在体外和体内与其自身的启动子结合，提示CaCRZ1表达的自调节机制。结论CaCrz1差异性结合其靶基因的启动子，以响应钙胁迫调节其表达。CaCrz1还通过其启动子中的5'-TGAGGGACTG-3'位调节其自身表达。录像摘要。启动子诱变表明，这两个CaCrz1绑定基序在CaUTR2表达的调节中起附加作用。此外，CaCRZ1基因受到CaCrz1的正调控。CaCrz1可以在体外和体内与其自身的启动子结合，提示CaCRZ1表达的自调节机制。结论CaCrz1差异性结合其靶基因的启动子，以响应钙胁迫调节其表达。CaCrz1还通过其启动子中的5'-TGAGGGACTG-3'位点调节其自身表达。录像摘要。启动子诱变表明，这两个CaCrz1结合基序在CaUTR2表达的调节中起附加作用。此外，CaCRZ1基因受到CaCrz1的正调控。CaCrz1可以在体外和体内与其自身的启动子结合，提示CaCRZ1表达的自调节机制。结论CaCrz1差异性结合其靶基因的启动子，以响应钙胁迫调节其表达。CaCrz1还通过其启动子中的5'-TGAGGGACTG-3'位调节其自身表达。录像摘要。结论CaCrz1差异性结合其靶基因的启动子，以响应钙胁迫调节其表达。CaCrz1还通过其启动子中的5'-TGAGGGACTG-3'位调节其自身表达。录像摘要。结论CaCrz1差异性结合其靶基因的启动子，以响应钙胁迫调节其表达。CaCrz1还通过其启动子中的5'-TGAGGGACTG-3'位调节其自身表达。录像摘要。