To persist in their dynamic human host environments, fungal pathogens must sense and adapt by modulating their gene expression to fulfill their cellular needs. Understanding transcriptional regulation on a global scale would uncover cellular processes linked to persistence and virulence mechanisms that could be targeted for antifungal therapeutics. Infections associated with the yeast Candida albicans, a highly prevalent fungal pathogen, and the multiresistant related species Candida auris are becoming a serious public health threat. To define the set of a gene regulated by a transcriptional regulator in C. albicans, chromatin immunoprecipitation (ChIP)-based techniques, including ChIP with microarray technology (ChIP-chip) or ChIP-DNA sequencing (ChIP-seq), have been widely used. Here, we describe a new set of PCR-based micrococcal nuclease (MNase)-tagging plasmids for C. albicans and other Candida spp. to determine the genome-wide location of any transcriptional regulator of interest using chromatin endogenous cleavage (ChEC) coupled to high-throughput sequencing (ChEC-seq). The ChEC procedure does not require protein-DNA cross-linking or sonication, thus avoiding artifacts related to epitope masking or the hyper-ChIPable euchromatic phenomenon. In a proof-of-concept application of ChEC-seq, we provided a high-resolution binding map of the SWI/SNF chromatin remodeling complex, a master regulator of fungal fitness in C. albicans, in addition to the transcription factor Nsi1 that is an ortholog of the DNA-binding protein Reb1 for which genome-wide occupancy was previously established in Saccharomyces cerevisiae. The ChEC-seq procedure described here will allow a high-resolution genomic location definition which will enable a better understanding of transcriptional regulatory circuits that govern fungal fitness and drug resistance in these medically important fungi.

中文翻译：

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念珠菌属的高分辨率全基因组占有率。使用ChEC-seq

为了在动态的人类宿主环境中持续存在，真菌病原体必须通过调节基因表达来感知和适应，以满足它们的细胞需求。了解全球范围内的转录调控将揭示与持久性和毒力机制相关的细胞过程，这些机制可以作为抗真菌治疗的目标。与酵母白色念珠菌（一种高度流行的真菌病原体）和多重耐药相关物种耳念珠菌相关的感染正在成为严重的公共健康威胁。定义由白色念珠菌中的转录调节因子调控的基因组，基于染色质免疫沉淀 (ChIP) 的技术，包括采用微阵列技术的 ChIP (ChIP-chip) 或 ChIP-DNA 测序 (ChIP-seq)，已被广泛使用。在这里，我们描述了一组新的基于 PCR 的微球菌核酸酶 (MNase) 标记质粒，用于白色念珠菌和其他念珠菌属 使用染色质内源切割 (ChEC) 与高通量测序 (ChEC-seq) 结合来确定任何感兴趣的转录调节因子的全基因组位置。ChEC 程序不需要蛋白质-DNA 交联或超声处理，从而避免了与表位掩蔽或超 ChIPable 常染色现象相关的伪影。在 ChEC-seq 的概念验证应用中，我们提供了 SWI/SNF 染色质重塑复合物的高分辨率结合图，它是白色念珠菌真菌适应性的主要调节因子，此外还有转录因子 Nsi1 DNA 结合蛋白 Reb1 的直向同源物，之前在酿酒酵母中建立了全基因组占有率. 此处描述的 ChEC-seq 程序将允许高分辨率基因组位置定义，这将有助于更好地了解控制这些医学上重要真菌中真菌适应性和耐药性的转录调控回路。