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

M. Masuda, D. Dulon, K. Pak, L.M. Mullen, Y. Li, L. Erkman, A.F. Ryan
Neuroscience. 2011-12-01; 197: 48-64
DOI: 10.1016/j.neuroscience.2011.09.033

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1. Neuroscience. 2011 Dec 1;197:48-64. doi: 10.1016/j.neuroscience.2011.09.033. Epub
2011 Sep 19.

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

Masuda M(1), Dulon D, Pak K, Mullen LM, Li Y, Erkman L, Ryan AF.

Author information:
(1)Department of Surgery/Otolaryngology, VA Medical Center and UCSD School of
Medicine, 9500 Gilman Drive 0666, La Jolla, CA 92093-0666, USA.

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.

Published by Elsevier Ltd.

DOI: 10.1016/j.neuroscience.2011.09.033
PMCID: PMC3243488
PMID: 21958861 [Indexed for MEDLINE]

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