Transcriptional regulation of the budding yeast DDI2 gene in response to environmental stresses

芽殖酵母 DDI2 基因响应环境胁迫的转录调控

• 批准号: RGPIN-2019-05604
• 负责人: Xiao, Wei
• 金额: $ 3.64万
• 依托单位: University of Saskatchewan
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744342
• 关键词: Transcriptional; regulation; budding; yeast; DDI2

DDI2 and DDI3 are two duplicated budding yeast DNA-damage inducible (DDI) genes identified through our microarray analysis. During the current granting period, we demonstrated that DDI2 encodes a HD-domain-containing cyanamide (CY) hydrotase, and determined the recombinant Ddi2 protein structure and its detailed mechanism of reaction. More interestingly, the DDI2/3 gene was induced by CY by up to 2,000-fold, and deletion of both DDI2 and DDI3 genes sensitizes cells to CY and MMS. Through a genome-wide genetic screen, we have identified a transcriptional factor Fzf1 that is absolutely required for the induction of DDI2 by CY and MMS. However, how DDI2 is induced by CY and MMS remains largely unknown. This proposed research is to characterize molecular mechanisms by which budding yeast DDI2 is induced in response to CY and MMS, and to investigate the stress responsive network mediated by Fzf1. (1) Define cis-acting element(s) required for the DDI2 . A DDI2 promoter-lacZ fusion construct is able to support full induction by CY and MMS. We will make DDI2 promoter deletions and subsequently point mutations in this construct and test their effects on the lacZ reporter expression to define the critical cis-acting element. This sequence will be used as a probe to perform electrophoresis mobility shift assay (EMSA) in vivo and in vitro to see if it is the Fzf1-binding site. If other cis-acting elements are also defined through the above analysis, we will search for their cognate transcriptional regulators by bioinformatics analysis and yeast one-hybrid screen. (2) Investigate how cells sense CY-induced stress. Upon CY or MMS treatment, the Fzf1 level was not significantly altered, nor was it modified by means like phosphorylation or ubiquitination. Based on our preliminary analysis, we are entertaining three alternative but non-exclusive hypotheses: (i) Certain amino acid residues in Fzf1 itself are modified by treatment with CY or MMS, which turns it into an active form; (ii) a histone subunit is modified by treatment with CY or MMS that alters nucleosome structure, allowing access of Fzf1 to the DDI2 promoter; and (iii) an unknown co-factor is modified by treatment with CY or MMS that recruits Fzf1 to the DDI2 promoter. (3) Structure and functions of Fzf1 in coordinating multiple chemical stress response pathways. Fzf1 appears to coordinate cellular response pathways to several chemical stresses. To further address how Fzf1 coordinately regulates these pathways, we will purify the Fzf1 protein and determine its crystal structure bound to the consensus DNA sequence and functional domains. Meanwhile, we will assess global transcriptional regulatory network by Fzf1 through RNA-seq analysis. The clustered genes will be further characterized to see if they are coordinately regulated in response to several chemical stresses including CY, MMS, sulphite and nitrogen oxide

DDI 2和DDI 3是通过我们的微阵列分析鉴定的两个重复的芽殖酵母DNA损伤诱导（DDI）基因。在目前的授权期间，我们证明了DDI 2编码一个含HD结构域的氰胺（CY）氢化酶，并确定了重组DDI 2蛋白的结构和其详细的反应机制。更有趣的是，DDI 2/3基因被CY诱导高达2,000倍，并且DDI 2和DDI 3基因的缺失使细胞对CY和MMS敏感。通过全基因组的遗传筛选，我们已经确定了一个转录因子Fzf 1是绝对需要的诱导DDI 2的CY和MMS。然而，CY和MMS如何诱导DDI 2仍然是未知的。本研究旨在探讨芽殖酵母DDI 2在CY和MMS诱导下的分子机制，以及Fzf 1介导的应激反应网络。(1)定义DDI 2所需的顺式作用元件。DDI 2启动子-lacZ融合构建体能够支持CY和MMS的完全诱导。我们将在此构建体中进行DDI 2启动子缺失和随后的点突变，并测试它们对lacZ报告基因表达的影响，以确定关键的顺式作用元件。该序列将用作探针，在体内和体外进行电泳迁移率变动试验（EMSA），以确定其是否为Fzf 1结合位点。如果通过上述分析还确定了其他的顺式作用元件，我们将通过生物信息学分析和酵母单杂交筛选寻找它们的同源转录调控因子。(2)研究细胞如何感知CY诱导的应激。在CY或MMS处理后，Fzf 1水平没有显著改变，也没有通过磷酸化或泛素化等方式进行修饰。基于我们的初步分析，我们考虑了三个可供选择但非排他性的假设：（i）Fzf 1本身的某些氨基酸残基通过CY或MMS处理而被修饰，这使其变成活性形式;（ii）组蛋白亚基通过CY或MMS处理而被修饰，这改变了核小体结构，允许Fzf 1进入DDI 2启动子;和（iii）通过用CY或MMS处理修饰未知辅因子，其将Fzf 1募集到DDI 2启动子。(3)Fzf 1在协调多种化学应激反应途径中的结构和功能。fzf 1似乎协调细胞对几种化学应激的反应途径。为了进一步解决Fzf 1如何协调调节这些途径，我们将纯化Fzf 1蛋白，并确定其与共有DNA序列和功能结构域结合的晶体结构。同时，我们将通过RNA-seq分析评估Fzf 1的全球转录调控网络。将进一步表征成簇的基因，以观察它们是否响应于几种化学胁迫（包括CY、MMS、亚硫酸盐和氮氧化物）而协同调节