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

• Published in: Neuroscience Vol. 197; pp. 48 - 64
• Main Authors: Masuda, M, Dulon, D, Pak, K, Mullen, L.M, Li, Y, Erkman, L, Ryan, A.F
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Ltd 01.12.2011; Elsevier
• Subjects: Animals; ATOH1; autoregulation; Basic Helix-Loop-Helix Transcription Factors - genetics; bioinformatics; Biological and medical sciences; Cell Line; Chromatin Immunoprecipitation; DNA - genetics; E-Box Elements - genetics; Fundamental and applied biological sciences. Psychology; gene expression; Gene Expression Regulation - genetics; hair cell; Hair Cells, Auditory, Inner; Homeodomain Proteins - biosynthesis; Homeodomain Proteins - genetics; Mice; Mice, Transgenic; Neurology; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; POU4F3; Transcription Factor Brn-3C - biosynthesis; Transcription Factor Brn-3C - genetics; Transfection; Vertebrates: nervous system and sense organs; transcription factor; basic helix-loop-helix; beta-galactosidase; p5; PFA; ChIP; bHLH; chromatin immunoprecipitation; red fluorescent protein; SG; paraformaldehyde; RFP; ATOH1; polymerase chain reaction; organ of Corti; eGFP; postnatal day 5; hair cell; autoregulation; POU4F3; hair cells; spiral ganglion; TF; enhanced green fluorescent protein; oC; bioinformatics; HC; gene expression; PCR; β-Gal; Rodentia; Ciliated cell; Gene expression; Vertebrata; Mammalia; Mouse; Animal; DNA; Self regulation
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/j.neuroscience.2011.09.033

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.