本文選題：POLD2 + FEN1　； 參考：《中國農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：DNA復(fù)制是生命最基本的進(jìn)程,細(xì)胞在分裂時,將遺傳信息DNA序列以及表觀遺傳信息如DNA甲基化、組蛋白修飾等精確的傳遞到下一代需要DNA復(fù)制元件的參與,以保證基因組的穩(wěn)定性。DNA復(fù)制的紊亂導(dǎo)致細(xì)胞DNA復(fù)制以及損傷脅迫的增加,引起基因組穩(wěn)定性降低,甚至導(dǎo)致癌癥的發(fā)生。在擬南芥中,對于DNA復(fù)制相關(guān)因子研究的相對較少,尤其是DNA復(fù)制因子如何參與表觀遺傳調(diào)控。本研究利用一個轉(zhuǎn)基因系統(tǒng)篩選調(diào)控轉(zhuǎn)錄水平基因沉默的因子,獲得了兩個組蛋白修飾因子]HDA6.UBP26和三個DNA復(fù)制因子RFC1、POLD2和FEN1。由于POLD2和FEN1在擬南芥中的功能研究的不是很清楚,因此本研究集中在這兩個基因上。通過亞細(xì)胞定位以及組織表達(dá)模式分析發(fā)現(xiàn)POLD2定位于細(xì)胞核并且在各個組織中均有表達(dá)。利用免疫沉淀結(jié)合質(zhì)譜分析表明POLD2可以同POLD1、POLD3、POLD4以及REV3共純化。遺傳分析發(fā)現(xiàn)POLD2同Polμα以及ATR協(xié)同調(diào)控植物的發(fā)育。另外，，POLD2突變導(dǎo)致基因組不穩(wěn)定,例如同源重組頻率的增加、對DNA損傷試劑敏感以及端粒的長度變短。細(xì)胞周期因子CYCB1;1的表達(dá)在pold2-1突變體中升高。在表觀遺傳學(xué)方面,全基因組DNA甲基化測序分析表明POLD2突變對DNA甲基化影響較小。進(jìn)一步進(jìn)行了全基因組組蛋白修飾的分析,包括H3K27me3,H3K4me3以及H3K9me2的ChIP-seq實驗。ChIP-seq和RNA-seq結(jié)果表明POLD2突變導(dǎo)致許多位點H3K27me3以及H3K4me3的變化,從而導(dǎo)致基因表達(dá)的異常。FEN1在DNA復(fù)制活躍的組織表達(dá)較多,比如根尖以及頂端分生組織。FEN1-GFP在煙草表皮細(xì)胞中定位于細(xì)胞核,在核仁有大量的富集。fen]-1突變體對DNA損傷試劑MMS超級敏感,同時也表現(xiàn)出端粒長度變短的表型。全基因組ChIP-seq和RNA-seq結(jié)果表明,FENl同POLD2一樣可以調(diào)控一部分基因的H3K27me3,從而影響基因的表達(dá)。綜上,本研究系統(tǒng)的研究了擬南芥兩個DNA復(fù)制相關(guān)基因POLD2和FENl。這兩個基因的突變導(dǎo)致了基因組穩(wěn)定性的下降,包括對DNA損傷試劑敏感以及端粒長度的變短。另外結(jié)合全基因組RNA-seq以及組蛋白修飾的分析揭示了DNA復(fù)制相關(guān)因子在維持H3K27me3的修飾以及基因沉默中起著至關(guān)重要的作用。本文的工作為探究DNA復(fù)制同基因組穩(wěn)定性以及表觀調(diào)控提供了一個新的線索。
[Abstract]:DNA replication is the most basic process of life. During cell division, DNA sequences and epigenetic information such as DNA methylation and histone modification are transferred to the next generation with the participation of DNA replication elements. In order to ensure the stability of genome, the disorder of DNA replication leads to the increase of DNA replication and damage stress, which leads to the decrease of genome stability and even the occurrence of cancer. In Arabidopsis thaliana, there are relatively few studies on DNA replication-related factors, especially how DNA replicators participate in epigenetic regulation. In this study, two histone modifiers HDA6.UBP26 and three DNA replicators RFC1PPOLD2 and FEN1 were obtained by screening genes silencing at transcriptional level by a transgenic system. Since the functional studies of POLD2 and FEN1 in Arabidopsis thaliana were not very clear, this study focused on these two genes. It was found that POLD2 was located in the nucleus and expressed in all tissues by subcellular localization and tissue expression pattern analysis. The results of immunoprecipitation and mass spectrometry showed that POLD2 could be co purified with POLD1, POLD3, POLD4 and REV3. Genetic analysis showed that POLD2 coordinated with Pol 渭 偽 and ATR to regulate plant development. In addition, POLD2 mutation leads to genomic instability, such as increased homologous recombination frequency, sensitivity to DNA damage reagents and shorter telomere length. The expression of cell cycle factor CYCB1-1 was increased in pold2-1 mutants. In epigenetics, genomic DNA methylation sequencing showed that POLD2 mutation had little effect on DNA methylation. Further analysis of genomic histone modification was carried out, including H3K27me3H3K4me3 and H3K9me2 ChIP-seq experiment. ChIP-seq and RNA-seq results showed that POLD2 mutation led to changes in H3K27me3 and H3K4me3. Thus, the abnormal expression of gene. FEN1 is more expressed in the tissues where DNA replication is active, such as root tip and apical meristem. FEN1-GFP is located in the nucleus of tobacco epidermal cells. A large number of enriched .fen] -1 mutants in nucleoli are super sensitive to DNA damage reagent MMS and also exhibit a shorter telomere length phenotype. The results of ChIP-seq and RNA-seq showed that FENl could regulate some genes H3K27me3 as POLD2, thus affecting gene expression. In conclusion, two DNA replication-related genes POLD2 and FENl. were systematically studied in Arabidopsis thaliana. Mutations in these two genes led to a decline in genomic stability, including sensitivity to DNA damage reagents and shorter telomere lengths. In addition, the analysis of RNA-seq and histone modification revealed that DNA replication-related factors play an important role in maintaining H3K27me3 modification and gene silencing. This work provides a new clue to explore the genomic stability and epigenetic regulation of DNA replication.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q943.2


本文編號：2066701