Molecular mechanisms of integration site selection by Dictyostelium retrotransposons

盘基网柄菌逆转录转座子整合位点选择的分子机制

• 批准号: 391682458
• 负责人: Professor Dr. Thomas Winckler
• 金额: --
• 依托单位: Lehrstuhl für Pharmazeutische Biologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/391682458?language=en
• 关键词: Molecular; mechanisms; integration; site; selection

Parasitism of genomes by mobile elements is a general phenomenon in biology. Although it is widely accepted that the activity of mobile elements may be beneficial for genome evolution of their hosts, there is also evidence that excessive amplification of selfish mobile elements may reduce host fitness by compromising genome stability. Thus, the study of interaction of mobile elements with their hosts genomes allows fundamental insights into genome evolution, the basis of organismal evolution.Especially in genomes with high gene density and reduced intergenic regions, mobile elements must evolve strategies to avoid insertional mutagenesis of host genes. Retrotransposons in the yeast Saccharomyces cerevisiae and the social amoeba Dictyostelium discoideum are intriguing examples of convergent evolution to solve this problem by targeting to the flanking regions of tRNA genes. Another means to avoid insertional mutagenesis of host genes, which is observed in genomes of plants, fungi and dictyostelid amoebae, seems to be the accumulation of mobile elements in gene-poor regions in heterochromatin and centromeric DNA. The D. discoideum genome accommodates retrotransposons of different phylogenetic origin that accumulate either in euchromatin in tRNA gene-flanking regions and in centromeric heterochromatin. Therefore, D. discoideum is an exceptional model to study different mechanisms of targeted integration by mobile elements and results obtained in this project will further expand the general knowledge about how dynamic parasite-host interactions contribute to genome evolution. In the first set of experiments we focus on retrotransposon TRE5-A. This element integrates with high precision upstream of tRNA genes in a presumed nucleosome-free region. Because TRE5-A integration depends on the host factor CbfA that contains a JmjC domain, a putative histone demethylase, we determine whether a CbfA-mediated nucleosome remodeling activity upstream of tRNA genes is required for position-specific integration of TRE5-A. In addition, we determine the RNA polymerase III transcription complex distribution in the D. discoideum genome to obtain direct proof that interaction with such complexes determines TRE5-A integration site selection. In the second set of experiments we compare the related DGLT-A and Skipper elements to evaluate what kind of interaction with chromatin determines integration at tRNA genes (DGLT-A) or in heterochromatin (Skipper). We extend preliminary data and determine whether interaction of the DGLT-A ribonuclease H domain with TFIIIC subunit Tfc4 determines integration site selection at tRNA genes in vivo. Further, we evaluate whether chromo domains in Skipper-like elements allow chromoviruses to integrate in heterochromatin, and whether functional degeneration of chromo domains may allow such elements to evolve new targeting strategies to colonize euchromatic regions of their hosts genomes.

移动的因子对基因组的寄生是生物学中的普遍现象。虽然人们普遍认为移动的元件的活性可能有利于其宿主的基因组进化，但也有证据表明，自私的移动的元件的过度扩增可能通过损害基因组稳定性而降低宿主适应性。因此，研究移动的元件与宿主基因组的相互作用，可以从根本上了解基因组进化，特别是在基因密度高、基因间隔区减少的基因组中，移动的元件必须进化出避免宿主基因插入突变的策略。在酵母酿酒酵母和社会变形虫Dictyosteelium discoideum中的反转录转座子是通过靶向tRNA基因的侧翼区域来解决这个问题的趋同进化的有趣例子。另一种避免宿主基因插入突变的方法是在异染色质和着丝粒DNA的基因缺失区积累移动的元件，这种方法在植物、真菌和网柄线虫的基因组中观察到。地方discoideum基因组容纳不同系统发育起源的逆转录转座子，这些逆转录转座子在tRNA基因侧翼区的常染色质和着丝粒异染色质中积累。因此，D. discoideum是一个特殊的模型，以研究不同的机制，有针对性的整合移动的元素和结果，在这个项目中获得的将进一步扩大一般知识的动态寄生虫-宿主相互作用如何有助于基因组进化。在第一组实验中，我们专注于反转录转座子TRE 5-A。该元件在假定的无核小体区域中以高精度整合在tRNA基因的上游。由于TRE 5-A的整合依赖于宿主因子CbfA，它含有一个JmjC结构域，一个假定的组蛋白去甲基化酶，我们确定是否需要一个CbfA介导的tRNA基因上游的核小体重塑活性的TRE 5-A的位置特异性整合。此外，我们还测定了RNA聚合酶III转录复合物在D. discoideum基因组获得直接证据，与这种复合物的相互作用决定了TRE 5-A整合位点的选择。在第二组实验中，我们比较了相关的DGLT-A和Skipper元件，以评估与染色质的何种相互作用决定了在tRNA基因（DGLT-A）或异染色质（Skipper）中的整合。我们扩展了初步的数据，并确定是否DGLT-A核糖核酸酶H结构域与TFIIIC亚基Tfc 4的相互作用决定在体内tRNA基因的整合位点选择。此外，我们评估是否染色体域在跳跃样元素允许染色病毒整合在异染色质，以及是否功能退化的染色体域可能允许这些元素发展新的靶向策略，以殖民其宿主基因组的常染色质区域。