Department of Biological Chemistry, School of Medicine, University of California, Irvine, Irvine, California 92697, USA.
School of Information and Computer Sciences, University of California, Irvine, Irvine, California 92697, USA.
Genome Res. 2019 Aug;29(8):1298-1309. doi: 10.1101/gr.240861.118. Epub 2019 Jun 27.
Retroelement integration into host genomes affects chromosome structure and function. A goal of a considerable number of investigations is to elucidate features influencing insertion site selection. The Ty3 retrotransposon inserts proximal to the transcription start sites (TSS) of genes transcribed by RNA polymerase III (RNAP3). In this study, differential patterns of insertion were profiled genome-wide using a random barcode-tagged Ty3. Saturation transposition showed that tRNA genes (tDNAs) are targeted at widely different frequencies even within isoacceptor families. Ectopic expression of Ty3 integrase (IN) showed that it localized to targets independent of other Ty3 proteins and cDNA. IN, RNAP3, and transcription factor Brf1 were enriched at tDNA targets with high frequencies of transposition. To examine potential effects of -acting DNA features on transposition, targeting was tested on high-copy plasmids with restricted amounts of 5' flanking sequence plus tDNA. Relative activity of targets was reconstituted in these constructions. Weighting of genomic insertions according to frequency identified an A/T-rich sequence followed by C as the dominant site of strand transfer. This site lies immediately adjacent to the adenines previously implicated in the RNAP3 TSS motif (CAA). In silico DNA structural analysis upstream of this motif showed that targets with elevated DNA curvature coincide with reduced integration. We propose that integration mediated by the Ty3 intasome complex (IN and cDNA) is subject to inputs from a combination of host factor occupancy and insertion site architecture, and that this results in the wide range of Ty3 targeting frequencies.
逆转录元件整合到宿主基因组中会影响染色体的结构和功能。相当多的研究旨在阐明影响插入位点选择的特征。Ty3 逆转录转座子插入 RNA 聚合酶 III（RNAP3）转录的基因的转录起始位点（TSS）附近。在这项研究中，使用随机条形码标记的 Ty3 对全基因组进行了插入差异模式的分析。饱和转座表明，即使在同功受体家族内，tRNA 基因（tDNAs）的靶向频率也有很大差异。Ty3 整合酶（IN）的异位表达表明，它与其他 Ty3 蛋白和 cDNA 独立地定位于靶标。IN、RNAP3 和转录因子 Brf1 在 tDNA 靶标中高度富集，转座频率较高。为了研究 - 作用 DNA 特征对转座的潜在影响，在具有限制数量的 5'侧翼序列加 tDNA 的高拷贝质粒上测试了靶向。在这些构建体中重建了靶标的相对活性。根据频率对基因组插入进行加权，确定了富含 A/T 的序列，其次是 C，作为链转移的主要位点。该位点紧邻先前涉及 RNAP3 TSS 基序（CAA）的腺嘌呤。在该基序上游的计算机 DNA 结构分析表明，具有升高 DNA 曲率的靶标与整合减少相吻合。我们提出，由 Ty3 intasome 复合物（IN 和 cDNA）介导的整合受到宿主因子占据和插入位点结构的组合输入的影响，这导致 Ty3 靶向频率的广泛范围。
