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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Basic Helix-Loop-Helix Transcription Factors / genetics*
  • Cell Line
  • Chromatin Immunoprecipitation
  • DNA / genetics
  • E-Box Elements / genetics*
  • Gene Expression Regulation / genetics*
  • Hair Cells, Auditory, Inner*
  • Homeodomain Proteins / biosynthesis
  • Homeodomain Proteins / genetics*
  • Mice
  • Mice, Transgenic
  • Oligonucleotide Array Sequence Analysis
  • Polymerase Chain Reaction
  • Transcription Factor Brn-3C / biosynthesis
  • Transcription Factor Brn-3C / genetics*
  • Transfection

Substances

  • Atoh1 protein, mouse
  • Basic Helix-Loop-Helix Transcription Factors
  • Homeodomain Proteins
  • Pou4f3 protein, mouse
  • Transcription Factor Brn-3C
  • DNA