Elsevier

Neuroscience

Volume 197, 1 December 2011, Pages 48-64
Neuroscience

Cellular and Molecular Neuroscience
Research Paper
Regulation of POU4F3 gene expression in hair cells by 5′ DNA in mice

https://doi.org/10.1016/j.neuroscience.2011.09.033Get rights and content

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 basic helix-loop-helix (bHLH) transcription factors, including motifs activated by ATOH1. Cotransfection of HEK293 or VOT-E36 cells with ATOH1 and the transgene as a reporter enhanced eGFP expression when compared with 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.

Highlights

▶A 5′ genomic DNA fragment from the murine Pou4f3 gene directs gene expression to hair cells. ▶The pattern of expression suggests multiple hair cell enhancers in the Pou4f3 gene. ▶Three regions within this fragment are highly conserved across mammals. ▶Clustered E-boxes are consistent with class II bHLH transcription factor binding. ▶The class II factor ATOH1 binds to, and regulates expression directed by, the 5′ DNA fragment.

Section snippets

Generation of transgenic mice

All procedures were approved by the Animal Subjects Committee of the San Diego VA Medical Center, and were in accordance with the National Institute of Health policies regarding the treatment of animal subjects.

An 8.5 kb genomic DNA fragment immediately 5′ to the ATG of the murine Pou4f3 gene was isolated from a strain 129 genomic library. The fragment was ligated to one of two reporter constructs. The first was a beta galactosidase (β-gal) coding sequence. The second consisted of an enhanced

Expression of β-gal was restricted to HCs within the inner ear of Pou4f3-8.5-β-gal mice

Three lines of Pou4f3-8.5-β-gal mice were generated and characterized in neonatal and adult inner ears. Within the inner ear, X-gal reaction product was restricted to HCs. In neonatal animals, all types of both cochlear and vestibular HCs exhibited β-gal expression. However, as illustrated in Fig 1a for cochlear HCs, the levels observed were highly variable, ranging from undetectable to highly dense. A similar response was observed in vestibular HCs (data not shown). Essentially the same

Discussion

Expression of eGFP under the control of 8.5 kb of 5′ Pou4f3 genomic DNA largely though not completely mimics, in transgenic mice, in vivo expression of Pou4f3 mRNA (Erkman et al., 1996, Artinger et al., 1998, Leonard et al., 2002). That is, expression occurred in most cells that normally produce POU4F3. Variable X-gal reaction product in the HCs of Pou4f3-8.5-β-gal mice seems likely to have resulted from irregular X-gal penetration into HCs, since in the eGFP transgenic, eGFP was uniformly

Acknowledgments

Supported by grants from the Research Service of the Veterans Administration, the NIH/NIDCD (RO1 DC000139), and the National Organization for Hearing Research (NOHR). Eduardo Chavez managed the mouse colony, Julie Lightner formatted the manuscript and Dr. Elizabeth Keithley read the manuscript and provided critical comments. Dr. Matthew Holley generously provided VOT-E36 cells. Their assistance is gratefully acknowledged.

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