Regulation of POU4F3 gene expression in hair cells by 5′ DNA in mice

Abstract

The POU-domain transcription POU4F3 is expressed in the sensory cells of the inner ear. Expression begins shortly after commitment to the hair cell (HC) fate, and continues throughout life. It is required for terminal HC differentiation and survival. To explore regulation of the murine Pou4f3 gene, we linked enhanced green fluorescent protein (eGFP) to 8.5 kb of genomic sequence 5’ to the start codon in transgenic mice. eGFP was uniformly present in all embryonic and neonatal HCs. Expression of eGFP was also observed in developing Merkel cells and olfactory neurons as well as adult inner and vestibular HCs, mimicking the normal expression pattern of POU4F3 protein, with the exception of adult outer HCs. Apparently ectopic expression was observed in developing inner ear neurons. On a Pou4f3 null background, the transgene produced expression in embryonic HCs which faded soon after birth both in vivo and in vitro. Pou4f3 null HCs treated with caspase 3 and 9 inhibitors survived longer than untreated HCs, but still showed reduced expression of eGFP. The results suggest the existence of separate enhancers for different HC types, as well as strong autoregulation of the Pou4f3 gene. Bioinformatic analysis of four divergent mammalian species revealed three highly-conserved regions within the transgene: 400 bp immediately 5’ to the Pou4f3 ATG, a short sequence at -1.3 kb, and a longer region at -8.2 to -8.5 kb. The latter contained E-box motifs that bind bHLH transcription factors, including motifs activated by ATOH1. Co-transfection of HEK293 or VOT-E36 cells with ATOH1 and the transgene as a reporter enhanced eGFP expression when compared to the transgene alone. Chromatin immunoprecipitation of the three highly conserved regions revealed binding of ATOH1 to the distal-most conserved region. The results are consistent with regulation of Pou4f3 in HCs by ATOH1 at a distal enhancer.

New treatment options for hearing loss

“…85), and its expression is necessary for the differentiation of hair cells during development 86 . Moreover, the transcription factors POU4F3 and GFI1, which are also necessary for differentiation and are needed to maintain the hair cell phenotype [87][88][89] , require ATOH1 for their expression [90][91][92] . The expression of ATOH1 in some cell types of the inner ear is sufficient to induce the formation of hair cells [93][94][95] .…”

Control of hair cell development by molecular pathways involving Atoh1, Hes1 and Hes5

“…Pou4f3 has several E-box motifs in one of its three highly conserved regions, which can be activated by ATOH1 in the inner ear hair cells (Masuda et al, 2011) (Table 1, Fig. 2).…”

“…Atoh1 is the first gene that was shown to be important in the inner ear hair cell development, acting as a "pro-hair cell gene" in the developing sensory epithelia (Bermingham et al, 1999). This gene interacts with other prosensory genes and regulates the expression of genes such as Pou4f3 (Masuda et al, 2011) and Gfi1 Yoon et al, 2011), which are significant in cell fate determination. Furthermore, the lateral inhibition of Atoh1 by Notch signaling and the downstream Hes1 and Hes5 are crucial for the determination of hair cells and supporting cells (Zheng et al, 2000;Lanford et al, 2000; reviewed by Bryant et al, 2002).…”

The regulation of gene expression in hair cells

“…Regulation of the pou4f3 gene in HCs was further evaluated by Masuda et al (2011) using a transgenic mouse model. They found that 8.5 kb of genomic DNA, 5′ to the initial codon of the pou4f3 coding sequence, was sufficient to drive expression of either beta galactosidase or GFP in HCs of the embryonic and neonatal inner ear (Fig.…”

“…Because the pou4f3 gene is expressed immediately after ATOH1 in HCs and given the data of Hu et al (2010) confirming upstream regulation, Masuda, et al (2011) evaluated the conserved regions of the transgene for ATOH1 preferred binding sites as defined by Klisch et al (2011). Three such sites were identified, two in the second distal conserved region, and one in the proximal conserved region (Fig.…”