Molecular mechanisms of integration site selection by Dictyostelium retrotransposons

可移动元素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，过度扩增自私的移动元件可能会损害基因组的稳定性，从而降低宿主的适合度。因此，研究可移动元件与宿主基因组的相互作用有助于深入了解基因组进化这一生物体进化的基础，特别是在基因密度高、基因间隔区少的基因组中，可移动元件必须进化策略以避免宿主基因的插入突变。酿酒酵母中的反转录转座子和盘状变形虫中的反转录转座子是通过靶向tRNA基因侧翼区域来解决这一问题的收敛进化的有趣例子。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中缺乏基因的区域积累可移动元件。盘状芽孢杆菌基因组具有不同起源的逆转录转座子，这些反转录转座子聚集在tRNA基因侧翼区的常染色质和着丝粒异染色质中。因此，盘状螺旋藻是研究移动元件靶向整合不同机制的一个例外模型，该项目的结果将进一步扩大关于动态寄生虫-宿主相互作用如何促进基因组进化的一般知识。在第一组实验中，我们重点研究了逆转录转座子TRE5-A。这个元件与tRNA基因上游的高精度整合在一个假定的核小体无性区。因为TRE5-A的整合依赖于含有JmjC结构域的宿主因子CBFA，组蛋白去甲基酶，我们确定CBFA介导的tRNA基因上游的核小体重塑活动是否是TRE5-A位置特异性整合所必需的。此外，我们还测定了RNA聚合酶III转录复合体在盘状芽孢杆菌基因组中的分布，以获得与此类复合体相互作用决定TRE5-A整合位点选择的直接证据。在第二组实验中，我们比较了相关的DGLT-A和Skipper元件，以评估什么样的与染色质的相互作用决定了tRNA基因(DGLT-A)或异染色质(Skipper)的整合。我们扩展了初步数据，并确定DGLT-A核糖核酸酶H结构域与TFIIIC亚基Tfc4的相互作用是否决定了体内tRNA基因的整合位点选择。此外，我们评估了Skipper-like元件中的染色域是否允许染色质病毒整合到异染色质中，以及染色域的功能性退化是否允许这些元件进化出新的靶向策略来定植其宿主基因组的常染色质区。

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.