A Tonotopic Regulatory Axis Governing Isoform-Specific MYO7A Expression in Cochlear Hair Cells

Abstract Myo7a, a gene mutated in Usher syndrome and non-syndromic deafness, encodes an unconventional myosin essential for hair cell function. Our previous work revealed that cochlear hair cells express distinct Myo7a isoforms with unique spatial and cell type-specific patterns. The canonical isoform (Myo7a-C) and a novel isoform (Myo7a-N) are co-expressed in outer hair cells (OHCs) but exhibit opposing tonotopic gradients, while inner hair cells (IHCs) primarily express Myo7a-C. These isoforms arise from distinct transcriptional start sites, indicating separate regulatory inputs. Here, we identify an intronic cis-regulatory element, EnhancerA, essential for tonotopically graded Myo7a expression. EnhancerA deletion reduces MYO7A protein levels in a tonotopically varied manner, disrupts hair bundle morphogenesis, alters OHC mechanotransduction, and leads to hair cell degeneration and hearing loss. We further identify SIX2, a tonotopically expressed transcription factor that may interact with EnhancerA to regulate Myo7a-N in OHCs. These findings define a cis-trans regulatory axis critical for isoform-specific Myo7a expression and cochlear function. Significance Cochlear hair cells rely on the molecular motor MYO7A for mechanosensory function. Our previous study revealed that Myo7a isoforms are differentially expressed in auditory hair cells, however, the mechanisms regulating this isoform-specific expression remain unclear. In this study, we identify a cis-regulatory element, EnhancerA, that governs MYO7A expression in a tonotopically graded manner. Furthermore, we demonstrate that the transcription factor SIX2 plays a role in regulation of MYO7A, and is essential for hair cell maintenance. These findings establish a mechanistic link between cochlear position and protein isoform diversity, highlighting how hair cells adapt to frequency-specific mechanical demands. Importantly, the tonotopic regulatory function of EnhancerA also offers new avenues for targeted gene therapy in auditory disorders. Competing Interest Statement The authors have declared no competing interest. Footnotes Competing interest statement: The Authors declare no competing interests Figure 3-6 revised. Updated results for Gfi1-cre and Myo15a-cre induced Six2 conditional knock-out mice.