探討酵母菌去甲基酶Rph1因應基因毒性逆境之轉錄調控機制

Mechanistic studies of the H3K36 demethylase Rph1 in transcriptional regulation responding to genotoxic stress in Saccharomyces cerevisiae

梁中怡(Chung-Yi Liang)

指導教授 ： 羅椀升

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.02971

摘要

基因表現決定了細胞的特性以及適度的生理反應，真核生物的DNA包裹在與組蛋白所纏繞成的核小體中，組蛋白上的後轉譯修飾能調控所有以DNA為模板的細胞生理活動。其中如何控制組蛋白的甲基化的動態平衡為基因調控重要的研究主題之一。本論文主要研究酵母菌中的組蛋白去甲基酶Rph1參與基因轉錄的調控機制。
本論文第一部份主要探討Rph1對於光解酶基因PHR1的轉錄抑制機制。Rph1可透過其去甲基酶活性而有效的抑制光解酶基因PHR1的轉錄，並利用其鋅手指結構結合到光解酶基因PHR1啟動子的上游抑制區域，並在核酸受損訊號活化之後離開PHR1啟動子，進而引發光解酶基因PHR1的轉錄；Rph1所導致的組蛋白去甲基化並能和轉譯抑制因子去乙醯基酶Rpd3一同合作達到組蛋白上甲基化與乙醯基化的交互作用，而抑制基因轉錄。檢查點蛋白Rad53能透過其激酶活性調控Rph1上的磷酸化修飾，改變Rph1結合染色質與否進而改變染色質構造來控制下游基因的轉錄。
第二部份針對Rph1調控標的進行全面性的分析。利用微陣列找出在剔除RPH1的突變株中差別表現的基因群，結果顯示Rph1在正常生長的細胞中主要功能為轉錄抑制因子，許多逆境相關基因其啟動子都含有Rph1辨識結合序列(CCCCTWA)，值得注意的是許多Rph1抑制的基因屬於”環境逆境共同反應基因”，受到各式各樣不同逆境共同引發表現的基因群，包含DNA損傷以及氧化壓力等等；Rph1可以直接結合到標的基因的啟動子進而抑制其表現，逆境可引發Rph1磷酸化修飾並且離開啟動子同時Rph1蛋白量也減少進而促使基因轉錄。
綜合而言，本論文發現了去甲基酶Rph1可經由兩種不同路徑調控基因表現：透過Rph1去甲基化酵素活性或透過Rph1直接結合到染色質，影響許多逆境相關基因。而在不同的環境底下，許多訊息傳遞路徑可能參與調控Rph1的磷酸化或是蛋白質表現量以及控制其染色質結合能力與否，來嚴密調節Rph1標的基因，適切反應不同環境的生理功能，以達到細胞正常生長的平衡。

關鍵字

染色質 ； 去甲基酶轉錄調控 ； 逆境反應

並列摘要

Alteration of gene expression is critical for cellular processes. The eukaryotic genome is wrapped with histone proteins to form a highly ordered chromatin structure. The post-translational modifications on histones provide pivotal regulations for DNA-templated events. The dynamics of histone methylation have emerged as an important issue since the identification of histone demethylases. This dissertation is focused on the mechanistic roles of the H3K36 demethylase Rph1 in transcriptional regulation.
First (Chapter II), we revealed that Rph1 is associated with the URS region of the PHR1 promoter via its zinc-finger domains and dissociated after UV irradiation. Rph1-mediated histone demethylation influences the dynamic cross-talk between histone methylation and acetylation in cooperation with the co-repressor Rpd3 on the promoter region. Furthermore, the checkpoint protein kinase Rad53 modulates Rph1 phosphorylation and dissociation leading to full activation of the PHR1 expression. Phosphorylation at S652 of Rph1 potentially contributes to its dissociation from chromatin and the transcriptional de-repression of PHR1 in response to DNA damage. H3K36 demethylase Rph1 regulates PHR1 expression by association with the promoter and by altering chromatin modifications under the control of DNA-damage checkpoint signaling.
Second (Chapter III), we explored the regulatory network of Rph1 in yeast by microarray analysis. More than 75% of Rph1-regulated genes showed increased expression in the rph1-deletion mutant, as compared with the wild type, suggesting that Rph1 is mainly a transcriptional repressor. The binding motif 5’- CCCCTWA -3’, which resembles the stress response element (STRE), is overrepresented in the promoters of Rph1-repressed genes. A significant proportion of Rph1-regulated genes respond to DNA damage and environmental stresses. Rad53 negatively modulated Rph1 protein level, which indicates a mechanistic role of Rad53 in the expression of Rph1-regulated genes. Furthermore, the results showed that Rph1 was a labile protein and the JmjN domain was important in maintaining protein stability and the repressive effect of Rph1. Rph1 is directly associated with the promoter region of targeted genes and dissociated from chromatin upon DNA damage and oxidative stress.
Here, we demonstrate Rph1 functions as a transcriptional repressor and an integral component connecting different signaling pathways responding to genotoxic stresses. The H3K36 demethylase activity and DNA binding affinity of Rph1 coordinately regulate the expression of target genes. Rph1 protein is highly dynamic under the control of multiple signaling pathways. Both DNA damage and oxidative stress induce Rph1 phosphorylation and dissociation from the promoters leading to transcriptional de-repression of Rph1 targets. Taken together, the delicate control of the H3K36 demethylase Rph1 not only increase the complexity of transcriptional regulation but also contributes to the maintenance of homeostasis and cell viability.

並列關鍵字

chromatin ； histone demethylase ； transcriptional regulation ； stress response

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