Investigating the Role of Lysine Demethylase 6B in Embryonic Development of Craniofacial Structure and Six1 Transcriptional Activity

Abstract

Branchio-oto-renal spectrum disorders (BOR) are autosomal genetic dominant disorders linked to craniofacial anomalies, primarily hearing loss, and renal defects. Variants of SIX1 or EYA1 account for a large portion of diagnosed cases, however the genetic causes of 15-45% of cases are unknown. Notably, patients carrying the same BOR-associated variant will present different degrees of defects (e.g., mild hearing loss with ear tags to profound hearing loss and hemifacial microsomia), suggesting other genetic modifiers can influence the severity of each case. Through yeast 2-hybrid and mass spectrometry-based (MS) proteomic screenings, Kdm6b was identified as a potential Six1-interacting protein. The goal of this study was to determine whether Kdm6b interacts with Six1 in vitro and in vivo in Xenopus laevis to characterize developmental defects that arise from Kdm6 inhibition. To determine whether kdm6b is expressed with six1 and its coactivator eya1 during craniofacial development, I generated an RNA probe to detect kdm6b mRNA expression and conducted whole-mount in situ hybridization (ISH) in Xenopus laevis at various embryonic developmental stages. I constructed an expression plasmid for Kdm6b to investigate if its activation influences Six1 transcriptional activity by luciferase assay. Additionally, we used a chemical inhibitor to observe dose-dependent morphological variations caused by Kdm6b inhibition in vivo. Findings from this work will characterize Kdm6b’s effect on Six1 transcriptional activity, establishing a foundation for future studies evaluating Kdm6b as a candidate gene involved in BOR.