Cadherin complexes recruit PIWIL2 to suppress transposons and maintain the normal epithelial phenotype - PROJECT SUMMARY Transposons, also known as transposable elements (TEs), are parasitic mobile DNA elements that, when re- activated, can introduce regulatory or protein coding changes at a new site in the genome. Previous studies showed TE activation was primarily in the germline and was thought to be overall inactive in differentiated somatic cells and tissues. However, transposon re-activation has been observed to occur frequently in the gastrointestinal tract, where it has been associated with diseases such as Crohn’s, colitis, and cancer. The reasons for increased TE activity in the gastrointestinal epithelium are still poorly understood, however. Interestingly, we have evidence of a novel interaction linking epithelial adherens junctions with transposon regulation. The adherens junctions, comprised of E-cadherin and p120 catenin as the core adhesion complex, act to stabilize cell-cell contacts and are frequently compromised in diseases of the colon epithelium. Preliminary studies in the lab have uncovered an interaction of the adherens junctions with PIWIL2 (PIWI-like mediated gene silencing 2). PIWIL2 is an endoribonuclease that is responsible for transposon silencing and germline maintenance by interacting with piRNAs (PIWI-interacting RNAs) to target TE RNA transcripts for degradation. We found co-localization of PIWIL2 with E-cadherin, p120 catenin, and PLEKHA7 which is an adherens junction specific protein marker in well-differentiated colon epithelial cells and tissues. Furthermore, our data show that disruption of the adherens junctions through depletion of E-cadherin and PLEKHA7 results in mis-localization of PIWIL2 to the cytoplasm or nucleus. E-cadherin depletion also increases the levels of the LINE1 transposon, which is the only autonomously active transposon in humans. Additionally, through RNA-sequencing, we have shown a specific subset of piRNAs are depleted when PLEKHA7 is depleted. From these data, we hypothesize that PLEKHA7 recruits PIWIL2 to the adherens junctions to suppress transposon activity in differentiated cells to maintain the integrity of the epithelium by preventing DNA damage caused by transposons. In Aim 1 we will examine how PLEKHA7 regulates the formation of a functional PIWIL2-piRNA complex, and use shRNA knockdown and CRISPR/Cas9 to deplete components of the complex and examine effects on its functionality by using retrotransposition and genomic integrity assays. The experiments proposed in Aim 2 will dissect the mechanism of PIWIL2 recruitment specifically to the mature apical adherens junctions in well-differentiated colon epithelial cells and examine whether disruption of this mechanism is critical in diseases such as inflammatory bowel disease (IBD), including Crohn’s and colitis, as well as colon cancer using patient tissues and an organoid system. Completion of the proposed study and training plan will further develop my technical skills, research independence, and clinical knowledge, and will position me to become a successful independent investigator.
