Expression of Pou3f3/Brn-1 and its genomic methylation in developing auditory epithelium.

In the mammalian cochlea, both the sensory cells-called hair cells (HCs)-and nonsensory cells such as supporting cells (SCs) and mesenchymal cells participate in proper auditory function through the expression of various functional molecules. During development, expression of certain genes is repressed through genomic methylation, one of the major epigenetic regulatory mechanisms. We explored the genomic regions that were differentially methylated in rat auditory epithelium at postnatal day 1 (P1) and P14 using amplification of intermethylated sites (AIMS). An AIMS fragment was mapped to the 3'-flanking region of Pou3f3/Brn-1. Bisulfite-converted PCR and quantitative methylation-specific PCR showed that the methylation frequency of the AIMS region and the adjacent CpG island was increased at P14, when the expression of Pou3f3 and the noncoding RNAs nearby decreased. Expression of de novo DNA methyltransferases 3a and 3b also suggests a role of epigenetic regulation during postnatal inner ear development. Immunohistochemical analysis showed that Pou3f3 was expressed specifically in the SCs and mesenchymal cells in the cochlea and established that Pou3f3 is a new cell-type marker for studying inner ear development. Mice deficient in Pou3f3 or Pou3f2 plus Pou3f3 did not exhibit any abnormality in the embryonic cochlea. Absence of Pou3f3 affected neither the proliferation nor the differentiation activities of HC progenitor cells. Pou3f3 may, however, be important for the maintenance or functional development of the postnatal cochlea. This is the first report to study involvement of an epigenetic regulatory mechanism in the developing mammalian auditory epithelium.