Ty Element Retrotransposition in Saccharomyces cerevisiae

我们的研究关注的是芽殖酵母中Ty元素逆转录的机制和后果。Ty元件包括五个相关的长末端重复（LTR）逆转录转座子家族，它们通过RNA中间体转座。Ty基因组包含两个与逆转录病毒Gag和Pol基因相对应的基因。逆转录转座子转录成基因组长度的RNA，该RNA是由元件编码的逆转录酶蛋白进行逆转录和翻译的模板。Ty蛋白成熟和逆转录发生在Ty病毒样颗粒（Ty- vlp）内，这似乎是转位过程所必需的。尽管Ty- vlps在细胞质中积累，但含有Ty cDNA的Ty预整合复合体，元件编码的整合酶和其他蛋白质必须通过核膜才能进入基因组。每个Ty元件类别非随机整合，并具有受染色质状态或RNA聚合酶III转录因子影响的独特靶向机制。所有现有的证据表明，Ty元素留在细胞内，不具有传染性。因此，这些元件及其宿主已经进化出控制机制，将转位和元件介导的基因组重排保持在较低水平，以及降低引起有害突变可能性的整合位点偏好。在过去的一年中，我们在表征调节Ty1反转录转位的宿主基因方面取得了进展。第一项研究包括对4739个基因缺失突变体进行系统筛选，以确定那些增加Ty1流动性（Ty1限制或RTT基因）的突变体。在鉴定的91个突变体中，80%编码的产物涉及染色质结构和功能、DNA修复和重组以及转录等核过程。然而，包含额外的Ty1和Ty3筛选的生物信息学分析表明，264个独特的基因参与各种生物过程，影响酵母中Ty的流动性。对我们筛选的33个rtt突变体的进一步表征表明，5个突变体的Ty1 RNA水平升高，其余突变体影响转录后的移动性。在转录延伸缺陷突变体（包括ckb2&#916；和elf1&#916；）中，Ty1 RNA和cDNA水平保持不变，这表明Ty1整合在这些菌株中可能更有效。CAN1位点的插入位点偏好需要Ty1限制性基因参与组蛋白H2B泛素化（Paf复合体亚基基因、BRE1和RAD6）、组蛋白H3乙酰化（RTT109和ASF1）和转录延伸（SPT5）。我们的研究结果表明，多种途径限制Ty1的迁移，组蛋白修饰可能保护编码区免受插入突变。由于这些基因也是RNA聚合酶II有效转录所必需的，因此停滞的转录复合体可能会发现Ty1插入的其他靶点。正在进行的工作集中在定义Ty1整合酶靶向区域和了解野生型和靶向缺陷突变体中转座事件的基因组景观。考虑到在各种筛选中发现的大量调节Ty反转录转位的基因，我们考虑了许多RTT基因通过一些共同途径起作用的可能性。最近与Joan Curcio的实验室（Wadsworth中心，纽约州奥尔巴尼）合作进行的一项关于Ty1限制性基因子集的研究明显支持了这一观点。出芽酵母中Ty1的迁移受到一系列蛋白质的限制，这些蛋白质在DNA复制和修复过程中起着保持基因组完整性的作用。然而，在缺乏这些Rtt因子（其中一些是哺乳动物逆转录病毒限制因子的同源物）的情况下，增加Ty1 cDNA水平和移动性的机制尚不清楚。有趣的是，在19个基因组保存缺陷的rtt突变体中，复制应激途径和DNA损伤途径这两种s期检查点途径部分或强烈地刺激了Ty1的迁移。相比之下，两个具有基因组维持能力的rtt突变体中，检查点途径都不参与激活Ty1。在rtt101&#8710突变体中，通过DNA损伤检查点蛋白Rad9、Rad24、Mec1、Rad53和Dun1刺激转位升高，而不是Chk1， Ty1编码的蛋白，而不是Ty1 cDNA，是检查点途径的直接靶点。在rtt101&#8710突变体中，Ty1整合酶和逆转录酶蛋白水平以及逆转录酶活性显著升高。我们假设，在缺乏基因组完整性因子的情况下产生的DNA损伤作为触发因素，通过s期检查点途径增强Ty1逆转录酶活性。

Our research concerns the mechanism and consequences of Ty element retrotransposition in the budding yeast Saccharomyces . Ty elements comprise five related families of long terminal repeat (LTR) retrotransposons that transpose via an RNA intermediate. The Ty genome contains two genes that correspond to the Gag and Pol genes of retroviruses. The retrotransposon is transcribed into a genome-length RNA, which is the template for reverse transcription by an element-encoded reverse transcriptase protein and for translation. Ty protein maturation and reverse transcription take place within Ty virus-like particles (Ty-VLPs), which appear to be essential for the transposition process. Although Ty-VLPs accumulate in the cytoplasm, a Ty preintegration complex containing Ty cDNA, the element-encoded integrase and perhaps other proteins must transit the nuclear membrane to gain access to the genome. Each Ty element class integrates nonrandomly and possesses distinctive targeting mechanisms that are influenced by the chromatin state or RNA polymerase III transcription factors. All available evidence suggests that Ty elements remain intracellular and are not infectious. Therefore, these elements and their host have evolved control mechanisms to keep transposition and element mediated genome rearrangements at a low level, and integration site preferences that reduce the possibility of causing deleterious mutations. Over the past year, we have made progress on characterizing host genes that modulate Ty1 retrotransposition. The first study involved a systematic screen of 4739 gene-deletion mutants to identify those that increase Ty1 mobility (Ty1 restriction or RTT genes). Among the 91 identified mutants, 80% encode products involved in nuclear processes such as chromatin structure and function, DNA repair and recombination, and transcription. However, bioinformatic analyses encompassing additional Ty1 and Ty3 screens indicate that 264 unique genes involved in a variety of biological processes affect Ty mobility in yeast. Further characterization of 33 of the rtt mutants identified in our screen show that Ty1 RNA levels increase in 5 mutants and the rest affect mobility posttranscriptionally. Ty1 RNA and cDNA levels remain unchanged in mutants defective in transcription elongation, including ckb2Δ and elf1Δ , suggesting Ty1 integration may be more efficient in these strains. Insertion site preference at the CAN1 locus requires Ty1 restriction genes involved in histone H2B ubiquitination by Paf complex subunit genes, as well as BRE1 and RAD6 , histone H3 acetylation by RTT109 and ASF1 , and transcription elongation by SPT5 . Our results indicate that multiple pathways restrict Ty1 mobility and histone modifications may protect coding regions from insertional mutagenesis. Since these genes are also required for efficient transcription by RNA polymerase II, additional targets for Ty1 insertion maybe uncovered by stalled transcription complexes. Ongoing work is focused on defining the Ty1 integrase targeting domain and understanding the genomic landscape available for transposition events in wild type and targeting-defective mutants. Considering the large number of genes identified in various screens that modulate Ty retrotransposition, we considered the possibility that many of the RTT genes act through a few common pathways. Support for this idea is evident from a recent study on a subset of Ty1 restriction genes, performed in collaboration with Joan Curcio's laboratory (Wadsworth Center, Albany NY). Mobility of Ty1 in budding yeast is restricted by an array of proteins that function to preserve the integrity of the genome during DNA replication and repair. However, the mechanisms involved in increasing Ty1 cDNA levels and mobility in the absence of these Rtt factors, several of which are orthologs of mammalian retroviral restriction factors, are poorly characterized. Interestingly, two S-phase checkpoint pathways, the replication stress pathway or the DNA damage pathway, partially or strongly stimulate Ty1 mobility in 19 rtt mutants with defects in genome preservation. In contrast, neither checkpoint pathway is involved in activating Ty1 in two rtt mutants that are competent for genome maintenance. In rtt101∆ mutants, in which elevated transposition is stimulated through DNA damage checkpoints proteins, Rad9, Rad24, Mec1, Rad53 and Dun1 but not Chk1, Ty1-encoded proteins, rather than Ty1 cDNA, are the direct targets of the checkpoint pathway. Levels of Ty1 integrase and reverse transcriptase proteins, as well as reverse transcriptase activity, are significantly elevated in rtt101∆ mutants. We hypothesize that DNA lesions created in the absence of genome integrity factors function as triggers that enhance Ty1 reverse transcriptase activity via S-phase checkpoint pathways.