The regulation of gene expression in hair cells

Abstract

No genes have been discovered for which expression is limited only to inner ear hair cells. This is hardly surprising, since the number of mammalian genes is estimated to be 20-25,000, and each gene typically performs many tasks in various locations. Many genes are expressed in inner ear sensory cells and not in other cells of the labyrinth. However, these genes are also expressed in other locations, often in other sensory or neuronal cell types. How gene transcription is directed specifically to hair cells is unclear. Key transcription factors that act during development can specify cell phenotypes, and the hair cell is no exception. The transcription factor ATOH1 is well known for its ability to transform nonsensory cells of the developing inner ear into hair cells. And yet, ATOH1 also specifies different sensory cells at other locations, neuronal phenotypes in the brain, and epithelial cells in the gut. How it specifies hair cells in the inner ear, but alternate cell types in other locations, is not known. Studies of regulatory DNA and transcription factors are revealing mechanisms that direct gene expression to hair cells, and that determine the hair cell identity. The purpose of this review is to summarize what is known about such gene regulation in this key auditory and vestibular cell type.

Fig. 1

Combinatorial regulation of GFP expression in the GER mediated by atoh1 enhancers (A), after co-transfection of SIX1, EYA1 and/or SOX2. Each embryonic GER was co-transfected with two reporter constructs; one in which a constitutive CMV promoter drove expression of red fluorescent protein (RFP) to identify all transfected cells, and another in which an atoh1 enhancer region was upstream from GFP. TF expression plasmids were also co-transfected at the same time. The proportion of yellow cells in the GFP/RFP images (given in %) represents the degree to which TF co-transfection induced GFP expression for each reporter. The 1.4 kb enhancer (B) was highly responsive to SIX1/EYA1, and this effect was enhanced by SOX2. Enhancer A (C) was moderately responsive to SOX2, and this effect was modestly enhanced by EYA1. Enhancer B was highly responsive to SIX1/EYA1 (D), but this effect was not influenced by SOX2 (not shown). From Ahmed et al. (2012), with permission.

Fig. 2

Postnatal expression of GFP in the inner ear of a transgenic mouse in which 8.5 kb of DNA 5′ to the start codon of the pou4f3 gene was used to drive expression of the reporter. a. All inner ear sensory epithelia express GFP. Within the cochlea (b–d) and vestibular labyrinth (e & f), only HCs express GFP. g. Phalloiden labeling (red) of GFP-positive HCs. Adapted from Masuda et al., 2012, with permission.

Fig. 3

Bioinformatic analysis of the 5′ region of the pou4f3 gene identified three homology regions (red) that are highly-conserved across four widely-separated mammalian species. The mouse sequence (8.5 kb) is illustrated. Highly conserved are a proximal region (Prox) immediately 5′ to the coding sequence and two distal regions (Dist I, Dist II) located up to 8.4 kb 5′. Within these regions, binding sites for 22 TFs that are expressed in the sensory epithelium of the embryonic inner ear at around the time of ATOH1 expression were also conserved between mouse, human, cow and dog. Question marks indicate short, highly-conserved sequences that do not correspond to known TF binding sites. Adapted from Ikeda et al. (2014), with permission.

Fig. 4

GFP and MYO7A expression in the GER induced by hATOH1 electroporation is enhanced by co-transfection with hNMYC. Transfection with hATOH1 alone induced ectopic GFP expression primarily in the GER. The array of native HCs is identified by arrows, and sequential imaging was used to identify any native HCs that might have migrated out of their normal positions during culture. Co-transfection of hATOH1 plus hNMYC enhanced induction of GFP in the GER by 68%. Moreover, the majority of GFP-positive cells were also immunopositive for MYO7A (arrows indicate a few GFP-positive cells that were negative for MYO7A), suggesting a broader adoption of a HC phenotype. Enhancement by NMYC is illustrated, but similar increases were also observed for GATA3, TCF3, ETC4 and ETS2. Adapted from Ikeda et al. (2014), with permission.

Fig. 5

a. Chromatin immunoprecipitation (ChIP) of TF DNA binding to conserved pou4f3 regions by TFs that enhance the ability of ATOH1 to induce GFP and MYO7A expression in cells of the early postnatal GER. TFs bound to the proximal (Prox) and/or second distal (Dist II) conserved regions of the 8.5 kb pou4f3 5′ sequence. No TFs bound to the first distal conserved region (Dist I), providing an internal PCR control. b. Semi-quantification of the PCR suggests especially strong relative binding of TFs to the second distal conserved region. Adapted from Ikeda et al. (2014), with permission.