Post-transcriptional modification in esophageal basal cells.

SUMMARY Balanced self-renewal and differentiation of basal cells is critical for the homeostasis and regeneration of the esophageal epithelium. Microenvironmental challenges including injury and allergy alter basal cell capability, leading to pathologies such as basal cell hyperplasia which is a key feature of eosinophilic esophagitis (EoE). We and others previously show that signaling pathways (e.g. BMPs) regulate the transcription of downstream targets to influence basal cells through transcription factors like p63. In contrast, little is known about how modifications of the resulting transcripts modulate basal cells. In this proposal we will fill the knowledge gap and provide initial insights into the mechanism by which posttranscriptional regulations modulate basal cell activities during homeosis and injury/repair. We will also investigate the role of posttranscriptional regulations in EoE basal cell hyperplasia. Specifically, we will study how Mettl3/14 complex-mediated RNA modifications are involved in these processes. Our central hypothesis is that Mettl3/14-mediated m⁶A deposition on transcripts including p63 is critical for maintaining basal cells and tissue integrity. We will test the hypothesis with three specific aims. Aim 1 will identify and determine the function of Mettl3/14-modified transcripts in basal cells. We will use a combination of mouse genetics, Nanopore sequencing, proteomics and CRSPR/Cas9 to identify and functionally test mRNA substrates that are modified by the Mettl3/14 complex. We will also study whether p63 transcripts are modified by the methylation complex. Aim 2 will determine the role of Mettl3/14 and p63 in basal cell-driven regeneration. We will use Mettl3 gain- and loss-of-function mouse models combined with p63 mutants to study how RNA modifications affect injury/repair of the esophagus. Aim 3 will test the hypothesis that inhibition of Mettl3/14 and p63 attenuates basal cell hyperplasia in an EoE mouse model. Together these studies will help us gain a better understanding of the normal mechanism regulating basal cell activities, meanwhile identifying new potential therapies for treating EoE.