PIWI - Transposon regulation by epithelial adherens junctions

项目摘要 转座子活性在超过50%的上皮癌中占基因组不稳定性。但 这一活动的原因尚不清楚。基于E-钙粘蛋白的粘附连接是重要的结构连接， 上皮细胞的组成部分，并经常在肿瘤中受损。我们发现钙粘蛋白 与PIWIL 2连接，PIWIL 2是负责沉默的piRNA加工途径的关键组分。 转座因子piRNA包括最大类别的小RNA，并且已经在许多领域中被广泛研究。 生殖系;然而，它们在体细胞组织中的作用尚不清楚。我们的初步数据显示， PIWIL 2在分化良好的乳腺、肾脏和结肠上皮细胞的成熟顶端粘附连接处， 而这种定位在癌细胞中丢失。有趣的是，E-cadherin的缺失导致了连接蛋白的丢失。 PIWIL 2的定位，在上调转座因子，和增加水平的γ-H2 AX，这是一个 DNA损伤指标。转座子活性增加的标志是DNA双链断裂。我们 假设粘附连接募集PIWIL 2以抑制分化的上皮细胞中的转座子活性， 细胞以维持基因组完整性和正常上皮表型。我们将在以下条件下检验这一假设： 以下目的：1）检查E-钙粘蛋白是否通过使转座子形成而抑制转座子水平和活性。 在分化良好的上皮细胞中的PIWI-piRNA复合物; 2）研究连接相关的 PIWIL 2抑制促肿瘤发生转化。这项工作意义重大，因为它将填补 PIWI转座子调节在分化的上皮组织和癌症中的作用的知识。的 该提议是创新的，因为它提供了细胞-细胞粘附、PIWI- 转座子生物学和基因组完整性。此外，它还采用了尖端技术，如皮尔纳- 转座子测序、CRISPR/Cas9基因组编辑和超分辨率显微镜。的长期目标 本研究旨在鉴定一种新的转座子沉默调节模式，由粘附连接协调， 这对于抑制转座子驱动的突变和肿瘤发生可能是关键的。成功 完成上述目标将有助于我们深入了解一种新的机制，这种机制将细胞结构与 基因组完整性，并为后续R 01级提案生成重要数据。

PROJECT SUMMARY Transposon activity accounts for genomic instability in more than 50% of epithelial cancers. However, the reasons of this activity are still unclear. The E-cadherin-based adherens junctions are essential structural components of the epithelial cells and frequently compromised in tumors. We have found association of cadherin junctions with PIWIL2, a key component of the piRNA-processing pathway that is responsible for silencing of transposable elements. piRNAs comprise the largest class of small RNAs and have been extensively studied in the germline; however, their roles in somatic tissues are unclear. Our preliminary data reveal localization of PIWIL2 at the mature apical adherens junctions of well-differentiated breast, kidney and colon epithelial cells, whereas this localization is lost in cancer cells. Interestingly, E-cadherin depletion results in loss of junctional localization of PIWIL2, in upregulation of a transposable element, and increased levels of γ-H2AX, which is an indicator of DNA damage. A hallmark of increased transposon activity is DNA double-stranded breaks. We hypothesize that the adherens junctions recruit PIWIL2 to suppress transposon activity in differentiated epithelial cells to maintain genomic integrity and the normal epithelial phenotype. We will test this hypothesis under the following Aims: 1) examine whether E-cadherin suppress transposon levels and activity by enabling formation of a PIWI-piRNA complex in well-differentiated epithelial cells; 2) investigate whether the junction-associated PIWIL2 suppresses pro-tumorigenic transformation. This work is significant, since it will fill a gap in the knowledge of the role of the PIWI-transposon regulation in differentiated epithelial tissues and in cancer. The proposal is innovative, since it provides an unexpected mechanistic link between cell-cell adhesion, PIWI- transposon biology and genomic integrity. In addition, it employs cutting-edge technologies, such as piRNA- transposon sequencing, CRISPR/Cas9 genome editing, and super-resolution microscopy. The long-term goal of this study is to identify a new mode of regulation of transposon silencing, coordinated by the adherens junctions, which could be critical for suppression of transposon-driven mutagenesis and tumorigenesis. Successful completion of the above Aims will help us gain insights into a new mechanism that tethers cell architecture to genomic integrity and generate significant data for subsequent R01-level proposals.