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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=715614
• 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是通过微阵列分析鉴定出的两个重复的芽殖酵母dna损伤诱导（DDI）基因。在目前的授权期内，我们证明了DDI2编码一个含hd结构域的氰酰胺（CY）氢化酶，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。通过全基因组遗传筛选，我们已经确定了一个转录因子Fzf1，它是CY和MMS诱导DDI2的绝对必需的。然而，DDI2是如何被CY和MMS诱导的在很大程度上仍然未知。本研究旨在研究出芽酵母DDI2响应CY和MMS的分子机制，并探讨Fzf1介导的胁迫响应网络。