SIX1 is required for adult cochlear sensory epithelium homeostasis and auditory function

The mammalian auditory sensory epithelium consists of luminal hair cells (HCs), whose stereociliary bundles convert sound vibrations into electrical signals, and basal supporting cells (SCs), which provide mechanical stability and homeostasis. During development, the transcription factor SIX1 specifies otic epithelial cells into neurosensory progenitors and commits progenitors to the HC lineage, but its contribution after birth has remained elusive. Here we show that SIX1 is essential for maintaining the structure and function of the sensory epithelium in the adult cochlea. Conditional deletion of Six1 in SCs, inner HCs, or outer HCs of 5–6-week-old mice results in profound deafness. SC-specific deletion causes rapid SC loss followed by secondary HC degeneration, whereas Six1 loss in inner or outer HCs disrupts stereociliary-bundle architecture and leads to progressive HC death. To uncover the underlying regulatory circuitry, we mapped genome-wide SIX1 occupancy in the mature cochlea. SIX1 binds DNA elements/enhancers near genes involved in cell adhesion, junctional integrity, actin dynamics, hair-bundle assembly, ion-channel, mechanotransduction, and cell survival, many of which are frequently mutated in hereditary deafness. Motif analysis showed that these SIX1-bound sites retain the core co-motifs found in SIX1 peaks in embryonic cochlea, suggesting reuse of embryonic cofactors. Notably, SIX1 occupies an intronic enhancer of Srf and physically interacts with SRF protein, positioning SIX1 atop an SRF-centered actin-cytoskeletal circuit crucial for bundle stability. Together, our findings redefine SIX1 as a lifelong regulator of cochlear homeostasis and reveal its downstream networks that maintain the adult auditory sensory epithelium.