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.

项目概要 转座子活性导致超过 50% 的上皮癌基因组不稳定。然而， 这项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 上皮细胞的组成部分，并且在肿瘤中经常受到损害。我们发现了钙粘蛋白协会 与 PIWIL2 的连接，PIWIL2 是 piRNA 加工途径的关键组成部分，负责沉默 转座元件。 piRNA 是最大的一类小 RNA，并已在以下领域进行了广泛研究： 种系；然而，它们在体细胞组织中的作用尚不清楚。我们的初步数据揭示了 PIWIL2位于分化良好的乳腺、肾和结肠上皮细胞的成熟顶端粘附连接处， 而癌细胞中则失去了这种定位。有趣的是，E-钙粘蛋白耗竭导致连接丧失 PIWIL2 的定位，转座元件的上调，以及 γ-H2AX 水平的增加，γ-H2AX 是一种 DNA损伤的指标。转座子活性增加的一个标志是 DNA 双链断裂。我们 假设粘附连接招募 PIWIL2 来抑制分化上皮细胞中的转座子活性 细胞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 以下目标：1) 检查 E-钙粘蛋白是否通过促进形成 分化良好的上皮细胞中的 PIWI-piRNA 复合物； 2）调查是否与连接相关 PIWIL2 抑制促肿瘤转化。这项工作意义重大，因为它将填补这一领域的空白 了解 PIWI 转座子调节在分化上皮组织和癌症中的作用。这 该提案具有创新性，因为它提供了细胞间粘附、PIWI-之间意想不到的机制联系 转座子生物学和基因组完整性。此外，它还采用了尖端技术，例如 piRNA- 转座子测序、CRISPR/Cas9 基因组编辑和超分辨率显微镜。长期目标是 这项研究旨在确定一种由粘附连接协调的转座子沉默调节的新模式， 这对于抑制转座子驱动的突变和肿瘤发生至关重要。成功的 完成上述目标将有助于我们深入了解将细胞结构与 基因组完整性并为后续 R01 级提案生成重要数据。