PIWI - Transposon regulation by epithelial adherens junctions

PIWI - 上皮粘附连接的转座子调节

• 批准号: 9979311
• 负责人: Antonis Kourtidis
• 金额: $ 20.97万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979311
• 关键词: Adherens Junction; Apical; Architecture; Area; Biological; Biological Assay; Biology; Breast; Breast Cancer Cell; Breast Epithelial Cells; CRISPR/Cas technology; Cadherins; Carcinoma; Cell Differentiation process; Cell Line; Cell-Cell Adhesion; Cells; Chemicals; Colon; Complex; DNA Damage; DNA Double Strand Break; DNA Transposable Elements; DNA Transposons; Data; Development; Disease; E-Cadherin; Elements; Epithelial; Epithelial Cells; Epithelium; Family; Foundations; Future; Gene Mutation; Genomic Instability; Genomics; Goals; Human Genome; Inherited; Kidney; Knowledge; Lead; Ligation; Link; Malignant Neoplasms; MicroRNAs; Microscopy; Modeling; Mutagenesis; Mutation; Normal tissue morphology; Oncogenes; Organ; Pathway interactions; Phenotype; RNA; RNA Interference; Radiation; Regulation; Research; Resolution; Retrotransposition; Role; Small RNA; Structure; Technology; Testing; Tissue Differentiation; Tissue Sample; Tissues; Tumor Promoters; Tumor Suppressor Proteins; Up-Regulation; Work; base; cancer cell; cell transformation; de novo mutation; genome editing; genome integrity; human tissue; innovation; insight; kidney epithelial cell; malignant breast neoplasm; mortality; novel; novel therapeutics; piRNA; prevent; recruit; targeted treatment; tool; transcriptome sequencing; transposon sequencing; tumor; tumorigenesis; tumorigenic

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.

项目总结 在超过50%的上皮性癌中，转座子活性是基因组不稳定的原因。然而， 这一活动的原因尚不清楚。基于E-钙粘附素的黏附连接是必需的结构 上皮细胞的组成部分，在肿瘤中经常受到损害。我们发现了钙粘附素的关联 与PIWIL2的连接，PIWIL2是负责沉默PIWIL2的piRNA加工途径的关键组成部分 转座因子。PiRNAs是小RNA中最大的一类，在 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本地化 在成熟的顶端，PIWIL2附着于分化良好的乳腺、肾脏和结肠上皮细胞的连接处， 然而，这种定位在癌细胞中丢失了。有趣的是，E-钙粘附素的耗尽会导致连接蛋白的丢失 PIWIL2的定位，上调了一个转座元件，并增加了γ-H2AX的水平，这是一种 DNA损伤的指示器。转座子活性增强的一个标志是DNA双链断裂。我们 假设黏附连接招募PIWIL2抑制分化上皮中转座子的活性 维持细胞基因组的完整性和正常的上皮表型。我们将在以下情况下测试这一假设 以下目的：1)研究E-钙粘附素是否通过促进转座子的形成抑制转座子的水平和活性 高分化上皮细胞中的PIWI-piRNA复合体；2)研究连接是否与 PIWIL2抑制促肿瘤转化。这项工作意义重大，因为它将填补 了解PIWI转座子调控在分化上皮组织和癌症中的作用。这个 这项提议是创新的，因为它提供了一种意想不到的细胞-细胞黏附之间的机械联系，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piRNA- 转座子测序、CRISPR/Cas9基因组编辑和超分辨显微镜。的长期目标是 本研究旨在确定一种新的调控转座子沉默的模式，该模式由附着连接协调， 这可能是抑制转座子驱动的突变和肿瘤形成的关键。成功 上述目标的完成将帮助我们深入了解一种新的机制，该机制将细胞结构捆绑到 基因组完整性，并为后续的R01级建议生成重要数据。