DNA复制因子POLD2和FEN1影响表观遗传以及基因组稳定性

发布时间：2018-06-25 16:21

  本文选题：POLD2 + FEN1　； 参考：《中国农业大学》2016年博士论文

【摘要】：DNA复制是生命最基本的进程,细胞在分裂时,将遗传信息DNA序列以及表观遗传信息如DNA甲基化、组蛋白修饰等精确的传递到下一代需要DNA复制元件的参与,以保证基因组的稳定性。DNA复制的紊乱导致细胞DNA复制以及损伤胁迫的增加,引起基因组稳定性降低,甚至导致癌症的发生。在拟南芥中,对于DNA复制相关因子研究的相对较少,尤其是DNA复制因子如何参与表观遗传调控。本研究利用一个转基因系统筛选调控转录水平基因沉默的因子,获得了两个组蛋白修饰因子]HDA6.UBP26和三个DNA复制因子RFC1、POLD2和FEN1。由于POLD2和FEN1在拟南芥中的功能研究的不是很清楚,因此本研究集中在这两个基因上。通过亚细胞定位以及组织表达模式分析发现POLD2定位于细胞核并且在各个组织中均有表达。利用免疫沉淀结合质谱分析表明POLD2可以同POLD1、POLD3、POLD4以及REV3共纯化。遗传分析发现POLD2同Polμα以及ATR协同调控植物的发育。另外，，POLD2突变导致基因组不稳定,例如同源重组频率的增加、对DNA损伤试剂敏感以及端粒的长度变短。细胞周期因子CYCB1;1的表达在pold2-1突变体中升高。在表观遗传学方面,全基因组DNA甲基化测序分析表明POLD2突变对DNA甲基化影响较小。进一步进行了全基因组组蛋白修饰的分析,包括H3K27me3,H3K4me3以及H3K9me2的ChIP-seq实验。ChIP-seq和RNA-seq结果表明POLD2突变导致许多位点H3K27me3以及H3K4me3的变化,从而导致基因表达的异常。FEN1在DNA复制活跃的组织表达较多,比如根尖以及顶端分生组织。FEN1-GFP在烟草表皮细胞中定位于细胞核,在核仁有大量的富集。fen]-1突变体对DNA损伤试剂MMS超级敏感,同时也表现出端粒长度变短的表型。全基因组ChIP-seq和RNA-seq结果表明,FENl同POLD2一样可以调控一部分基因的H3K27me3,从而影响基因的表达。综上,本研究系统的研究了拟南芥两个DNA复制相关基因POLD2和FENl。这两个基因的突变导致了基因组稳定性的下降,包括对DNA损伤试剂敏感以及端粒长度的变短。另外结合全基因组RNA-seq以及组蛋白修饰的分析揭示了DNA复制相关因子在维持H3K27me3的修饰以及基因沉默中起着至关重要的作用。本文的工作为探究DNA复制同基因组稳定性以及表观调控提供了一个新的线索。
[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 伪 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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2


本文编号：2066701