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Abstract

Homeostasis of the skin is achieved through tightly-regulated control of epidermal cell fate. Loss of such regulation will lead to various skin diseases and cancer. While the regulatory process has been studied over several decades, how the epidermal stem cells are fine-tuned to transit between different cell fates is not well understood. A better understanding of the mechanisms will provide us with better ways to enhance skin regeneration to help trauma or burn patients. Additionally, it will give us more insight in designing targeted agents against keratinocytic tumors of the skin. Many chemical modifications have been discovered in RNA, and methylation on the N6-adenoinse position (m6A) is one of the most prevalent modifications found in poly-adenylated RNAs, including messenger RNAs and long non-coding RNAs (lncRNAs). The “writer proteins”, METTL3 and METTL14, forms a methyltransferase complex that adds m6A to RNA, while the “eraser proteins”, FTO and ALKBH5 removes the methyl groups. Depending on the “reader protein”, m6A-modified RNAs can have various outcomes, including change in stability, splicing, localization, or RNA-protein interaction. RNA m6A modification has been found to play an essential role during embryonic development, and dysregulation of m6A modification protein have been found in various cancers, suggestive of its regulatory role in a wide range of tissues. This dissertation work is based on the central hypothesis that RNA m6A modification is an important regulator of epidermal stemness. We first investigated the effect of removing m6A modification by depleting the METTL14 in mouse skin. We found that loss of METTL14 leads to dramatic changes in the skin structure and impaired wound healing. In vitro assays also showed that the cells have clear stemness defects. Next, to determine the downstream effector of m6A modification, we performed m6A-sequencing comparing differentiated and undifferentiated epidermal progenitor cells. This revealed several lncRNAs with significant m6A changes. PVT1 is the only lncRNA with significantly reduced m6A level upon epidermal differentiation. We thus hypothesized that Pvt1 is the main regulatory target of m6A modification in epidermal cells. We next knocked out PVT1 in epidermal progenitor cells and found that the cells display stemness defects similar to that seen in METTL14 knockout. Pvt1 is known to interact with, and stabilizes MYC protein, and MYC is an important regulator of epidermal homeostasis. We thus hypothesized that m6A modified-Pvt1 can enhance its interaction with MYC. We found that the non-methylating PVT1 mutant, as well as PVT1 with m6A removed by FTO, has significantly reduced MYC interaction. In addition, lack of PVT1 interaction led to rapid degradation of MYC. Finally, we showed that by overexpressing MYC in METTL14 knockout epidermal cells, the stemness defect can be restored. In summary, this dissertation work reveals a novel role of m6A RNA modification in epidermal regulation. In brief, epidermal stem cells express higher level of m6A methyltransferases, which increases m6A modifications on Pvt1. Pvt1 with m6A modification were able to interact with MYC, allowing MYC to remain stable in the cells and maintain epidermal stemness.

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