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.