Function of IRF6 in regulating E-cadherin dependent adherens junctions

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PROJECT SUMMARY Over 70 million surgeries are performed in the United States annually. Although many wounds heal without problem, half require postsurgical wound care. Wounds that do not heal affect about 7 million people annually and generate treatment costs of about $100 billion, which creates a significant financial burden on the US economy. Increasing our understanding of the molecular pathways regulating wound healing would enhance tissue repair and reduce healthcare costs. Our long-term goal is to identify molecular pathways regulating tissue repair. We previously demonstrated that the transcription factor Interferon Regulatory Factor 6 (IRF6) is required for proper wound healing by acting as a master regulator of keratinocyte differentiation, proliferation, and collective cell migration. Our recent preliminary data show that Irf6-deficient keratinocytes have weaker cell-cell adhesion, and reduced membrane localization of adherens junction components, including E-cadherin, providing a potential rationale for the IRF6- dependent keratinocyte migration defect. Interestingly, our preliminary data also revealed that total adherens junction protein levels were not changed, suggesting a non-transcriptional function of IRF6 in these processes. IRF6, as a member of the Interferon regulatory transcription factor family, contains a highly conserved N- terminal, DNA-binding domain, and a less conserved protein interaction domain. Most of IRF6 described functions have been associated with its transcriptional activity, and very little is known about the functions of its protein interaction domain. Particularly, which domain of IRF6 contributes to cell-cell adhesions required for wound healing, is unknown. Our central hypothesis is that IRF6 promotes collective cell migration via a non- transcriptional regulation of cell-cell adhesion molecules at the cytoplasmic membrane. We will test our central hypothesis with the execution of two aims. In Aim 1 we will determine how IRF6 regulates E-cadherin trafficking. In Aim 2 we will determine how IRF6 promotes collective cell migration. To test our hypothesis, using a wide range of biochemical and cellular assays, we will take advantage of multiple IRF6 mutant lines to determine which domains of this transcription factor are required for regulating cell adhesions. The same mutant cell lines will be used to perform scratch wounds in 2D and excisional wounds in 3D models which will shed light on the importance of each domain of IRF6 in collective cellular migration. At the completion of this study, we will have identified a novel mechanism by which this transcription factor regulates vesicular trafficking necessary for cell adhesion organization, which could provide a molecular mechanism for the increased risk of surgical complications observed in patients with IRF6 mutations.