Transcriptional regulation of Atp2b2 determines the severity of AHL in C57BL/6J Mice

Thesis (Ph.D.)--University of Washington, 2015-12University of Washington Abstract Transcriptional regulation of Atp2b2 determines the severity of AHL in C57BL/6J Mice Rebecca R. Minich Chair of the Supervisory Committee: Professor Bruce L Tempel Otolaryngology-Head and Neck Surgery, Pharmacology Along with Na+/Ca2+ exchangers, plasma membrane Ca2+ ATPases (ATP2Bs) are main regulators of intracellular Ca2+ levels. Mouse mutations in Atp2b2 indicate that tight regulation of this gene is imperative for normal cell physiology, specifically in the auditory system. Additionally, mutations in Atp2b2 worsen hearing loss associated with mutations in the tip-link protein Cadherin 23 (CDH23) (Noben-Trauth et al. 2003; Schultz et al. 2005). This interaction is likely due to the necessity of Ca2+ in maintaining the structural integrity of the tip-links (Sotomayor et al. 2010). C57BL/6J (B6) mice have a severe age-related hearing loss (AHL) phenotype partially due to mutations in CDH23 (Noben-Trauth et al. 2003; Kane et al. 2012) . In this dissertation, we investigate whether Atp2b2 is another contributor to AHL of B6 mice in three parts: (I) The αAtp2b2 minimal promoter was identified to better understand transcriptional regulation of Atp2b2. Further characterization determined that ATOH1 and EGR1 are modulators of αAtp2b2 transcription in OC-1 and N2A cells. Shift assays identify a binding site for EGR1 in the minimal promoter of αAtp2b2 71 bases upstream of the transcriptional start site (TSS). (II) Two discreet measures of gene expression show misregulation of Atp2b2 transcript in B6 mice. We found that the proximal promoter of αAtp2b2 does not play a role in the misregulation of Atp2b2 transcript. However, two lncRNAs were identified in this region. Gene expression analysis and overexpression studies suggest that these lncRNAs inhibit expression of Atp2b2 transcript. This implicates misregulation of Atp2b2 by lnc83 in AHL of B6 mice. (III) To further investigate the contribution of Atp2b2 to B6 AHL, two congenic mice were generated: (1) B6 haplotype at Atp2b2 in a CB background (CB.B6Atp2b2) and (2) CB haplotype at Atp2b2 in a B6 background (B6.CBAtp2b2). We found that B6.CBAtp2b2 mice have an intermediate auditory phenotype at high frequencies and normal hearing at low frequencies. CB.B6Atp2b2 mice have a slight elevation of threshold at 5.6 kHz at 6 months of age. At 12 weeks of age, Atp2b2 expression is normal in brainstem tissue of B6.CBAtp2b2.and CB.B6Atp2b2 mice while lnc83 transcript levels are elevated in CB.B6Atp2b2mice. From this data we conclude that mutations in the Atp2b2 gene are necessary but not sufficient to cause the B6 AHL phenotype