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.
