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: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=738397
• 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

DDI2和DDI3是我们的微阵列分析中发现的两个重复的芽期酵母DNA损伤诱导基因。在本授权期内，我们证实了DDI2编码一个含HD结构域的氰胺(CY)水解酶，并确定了重组Ddi2蛋白的结构及其详细的反应机制。更有趣的是，DDI2/3基因被CY诱导了高达2000倍，DDI2和DDI3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CY和MMS诱导DDI2所必需的转录因子Fzf1。然而，CY和MMS如何诱导DDI2仍在很大程度上尚不清楚。本研究旨在研究CY和MMS诱导发芽酵母DDI2的分子机制，并研究Fzf1介导的胁迫响应网络。(1)定义DDI2所需的顺式作用要素(S)。构建的DDI2启动子-LacZ融合载体能够支持CY和MMS的完全诱导。我们将在该结构中进行DDI2启动子的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。该序列将被用作探针进行体内和体外的凝胶迁移率改变分析(EMSA)，以确定它是否是Fzf1结合位点。如果通过上述分析也定义了其他顺式作用元件，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诱导的应激反应。在CY或MMS处理后，Fzf1水平没有明显改变，也没有通过磷酸化或泛素化等手段进行修饰。根据我们的初步分析，我们考虑了三个可选但非排他性的假设：(I)Fzf1本身的某些氨基酸残基通过CY或MMS处理被修饰，使其成为一种活性形式；(Ii)组蛋白亚基通过CY或MMS处理而改变，从而改变核小体结构，允许Fzf1接近DDI2启动子；以及(Iii)未知辅助因子通过CY或MMS处理而被修改，该处理将Fzf1招募到DDI2启动子。(3)Fzf1在协调多种化学胁迫反应途径中的结构和功能。Fzf1似乎协调了细胞对几种化学胁迫的反应途径。为了进一步研究Fzf1是如何协调调节这些途径的，我们将纯化Fzf1蛋白，并确定其与一致的DNA序列和功能域结合的晶体结构。同时，我们将通过RNA-seq分析来评估Fzf1的全球转录调控网络。这些聚集的基因将被进一步鉴定，以确定它们是否对包括CY、MMS、亚硫酸盐和氮氧化物在内的几种化学胁迫做出协调调节