Function and regulation of Cph2 in Candida albicans [ChIP-Seq]. Candida albicans

Candida albicans is associated with humans as both a harmless commensal organism and a pathogen. Cph2 is a transcription factor whose DNA binding domain is similar to mammalian sterol response element binding proteins (SREBPs). SREBPs are master regulators of cellular cholesterol levels, and are highly conserved from fungi to mammals. However, ergosterol biosynthesis is regulated by the zinc finger transcription factor Upc2 in C. albicans and several other yeasts. Cph2 is not necessary for ergosterol biosynthesis, but important for colonization in the murine gastrointestinal tract. Here we demonstrate that Cph2 is a membrane-associated transcription factor that is processed to release the N-terminal DNA binding domain like SREBPs; but its cleavage is not regulated by cellular levels of ergosterol or oxygen. ChIP-Seq shows that Cph2 binds to the promoters of HMS1 and other components of the regulatory circuit for GI tract colonization. In addition, 50% of Cph2 targets are also bound by Hms1 and other factors of the regulatory circuit. Several common targets function at the head of the glycolysis pathway. Thus, Cph2 is an integral part of the regulatory circuit for GI colonization that regulates glycolytic flux. RNA-seq shows a significant overlap in genes differentially regulated by Cph2 and hypoxia, and Cph2 is important for optimal expression of some hypoxia-responsive genes in glycolysis and the citric acid cycle. We suggest that Cph2 and Upc2 regulate hypoxia-responsive expression in different pathways, consistent with a synthetic lethal defect of the cph2 upc2 double mutant in hypoxia. Overall design: Genome binding/occupancy profiling by high throughput sequencing. ChIP-seq of Cph2 was carried out in a wild-type strain carrying N-terminal myc-tagged Cph2 under the MAL2 promoter (MAL2-myc-Cph2N). IP and INPUT samples from 2 independent experiments, as well as a sample of untagged wild-type control, were sequenced.

白色念珠菌（Candida albicans）既可作为无害的共生微生物，也可作为致病菌与人类宿主形成关联。Cph2是一种转录因子，其DNA结合结构域与哺乳动物固醇反应元件结合蛋白（SREBPs）相似。SREBPs是细胞胆固醇水平的核心调控因子，在从真菌到哺乳动物的进化过程中高度保守。然而，在白色念珠菌及其他数种酵母中，麦角固醇的生物合成由锌指转录因子Upc2调控。Cph2并非麦角固醇生物合成所必需，但对小鼠胃肠道定植过程至关重要。

本研究证实，Cph2是一种膜结合型转录因子，可通过蛋白剪切释放N端DNA结合结构域，这一机制与SREBPs类似；但其剪切过程并不受细胞内麦角固醇或氧气水平的调控。染色质免疫共沉淀测序（ChIP-Seq）结果显示，Cph2可结合HMS1的启动子区域以及胃肠道定植调控通路的其他组分。此外，50%的Cph2靶基因同时可被Hms1及该调控通路的其他因子结合。部分共有靶基因定位于糖酵解通路的上游调控节点。因此，Cph2是调控糖酵解通量的胃肠道定植调控通路的核心组成部分。

RNA测序（RNA-seq）结果显示，受Cph2差异调控的基因与低氧应答基因存在显著重叠；Cph2对糖酵解与三羧酸循环中部分低氧应答基因的最佳表达至关重要。本研究表明，Cph2与Upc2通过不同通路调控低氧应答基因的表达，这与cph2 upc2双突变体在低氧环境下的合成致死缺陷表型一致。

实验整体设计：通过高通量测序开展全基因组结合/占据谱分析。本研究在携带MAL2启动子驱动的N端myc标签融合Cph2（MAL2-myc-Cph2N）的野生型菌株中开展Cph2的ChIP-seq实验。对2次独立实验得到的免疫沉淀（IP）与输入（INPUT）样本，以及未带标签的野生型对照样本进行了测序。