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Regulation of the orphan nuclear receptor Nr2f2 by the DFNA15 deafness gene Pou4f3.

Tornari C, Towers ER, Gale JE, Dawson SJ - PLoS ONE (2014)

Bottom Line: These sites were shown to be required for POU4F3 activation as their mutation leads to a reduction in the response of an Nr2f2 5' flanking region reporter construct to POU4F3.Immunocytochemistry was carried out in the developing and adult inner ear in order to investigate the relevance of this interaction in hearing.NR2F2 expression in the postnatal mouse organ of Corti was shown to be detectable in all sensory epithelia examined and characterised.

View Article: PubMed Central - PubMed

Affiliation: UCL Ear Institute, University College London, London, United Kingdom.

ABSTRACT
Hair cells are the mechanotransducing cells of the inner ear that are essential for hearing and balance. POU4F3--a POU-domain transcription factor selectively expressed by these cells--has been shown to be essential for hair cell differentiation and survival in mice and its mutation in humans underlies late-onset progressive hearing loss (DFNA15). The downstream targets of POU4F3 are required for hair cell differentiation and survival. We aimed to identify such targets in order to elucidate the molecular pathways involved in hair cell production and maintenance. The orphan thyroid nuclear receptor Nr2f2 was identified as a POU4F3 target using a subtractive hybridization strategy and EMSA analysis showed that POU4F3 binds to two sites in the Nr2f2 5' flanking region. These sites were shown to be required for POU4F3 activation as their mutation leads to a reduction in the response of an Nr2f2 5' flanking region reporter construct to POU4F3. Immunocytochemistry was carried out in the developing and adult inner ear in order to investigate the relevance of this interaction in hearing. NR2F2 expression in the postnatal mouse organ of Corti was shown to be detectable in all sensory epithelia examined and characterised. These data demonstrate that Nr2f2 is a direct target of POU4F3 in vitro and that this regulatory relationship may be relevant to hair cell development and survival.

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POU4F3-mediated activation of PRE1, PRE2 and a 4.2 kb Nr2f2 5′ flanking sequence.a. Schematic diagram of reporter constructs used in luciferase reporter assays in ND7 cells. POU4F3 recognition element (PRE) 1 and 2 are shown in an Nr2f2 5′ flanking region fragment (grey box) cloned upstream of a luciferase reporter gene (LUC). The location of this fragment relative to the Nr2f2 transcriptional start site is shown. b. Evaluation of the response of 4.2 kb-Nr2f2-Luc to increasing levels of POU4F3 or dreidel mutant POU4F3 (Ddl) expression construct. The luciferase activity of the reporter is normalised to its response to the empty expression vector and results are expressed relative to this. c. Response of the PRE1-Luc reporter construct in co-transfection experiments with POU4F3 and dreidel expression constructs. d. Evaluation of the response of PRE2-Luc in similar experiments to c. Error bars represent the s.e.m in b, c and d (n = 6 for each data point).
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pone-0112247-g002: POU4F3-mediated activation of PRE1, PRE2 and a 4.2 kb Nr2f2 5′ flanking sequence.a. Schematic diagram of reporter constructs used in luciferase reporter assays in ND7 cells. POU4F3 recognition element (PRE) 1 and 2 are shown in an Nr2f2 5′ flanking region fragment (grey box) cloned upstream of a luciferase reporter gene (LUC). The location of this fragment relative to the Nr2f2 transcriptional start site is shown. b. Evaluation of the response of 4.2 kb-Nr2f2-Luc to increasing levels of POU4F3 or dreidel mutant POU4F3 (Ddl) expression construct. The luciferase activity of the reporter is normalised to its response to the empty expression vector and results are expressed relative to this. c. Response of the PRE1-Luc reporter construct in co-transfection experiments with POU4F3 and dreidel expression constructs. d. Evaluation of the response of PRE2-Luc in similar experiments to c. Error bars represent the s.e.m in b, c and d (n = 6 for each data point).

Mentions: Having demonstrated the ability of POU4F3 to bind to PRE1 and PRE2, we tested whether POU4F3 can regulate the Nr2f2 5′ flanking region that contains these sites. A luciferase reporter construct containing 4.2 kb of the Nr2f2 5′ flanking region (4.2 kb-Nr2f2-Luc, Figure 2a) was used in co-transfection studies in ND7 cells. This analysis demonstrated a dose-dependent increase in 4.2 kb-Nr2f2-Luc activity in response to increasing POU4F3 levels. Compared to basal activity, promoter activity was five times higher at the maximal amount of POU4F3 expression construct used (3 µg) and was not replicated with a non-DNA-binding POU4F3 mutant (dreidel), showing that this activation is dependent on POU4F3-specific DNA binding (Figure 2b).


Regulation of the orphan nuclear receptor Nr2f2 by the DFNA15 deafness gene Pou4f3.

Tornari C, Towers ER, Gale JE, Dawson SJ - PLoS ONE (2014)

POU4F3-mediated activation of PRE1, PRE2 and a 4.2 kb Nr2f2 5′ flanking sequence.a. Schematic diagram of reporter constructs used in luciferase reporter assays in ND7 cells. POU4F3 recognition element (PRE) 1 and 2 are shown in an Nr2f2 5′ flanking region fragment (grey box) cloned upstream of a luciferase reporter gene (LUC). The location of this fragment relative to the Nr2f2 transcriptional start site is shown. b. Evaluation of the response of 4.2 kb-Nr2f2-Luc to increasing levels of POU4F3 or dreidel mutant POU4F3 (Ddl) expression construct. The luciferase activity of the reporter is normalised to its response to the empty expression vector and results are expressed relative to this. c. Response of the PRE1-Luc reporter construct in co-transfection experiments with POU4F3 and dreidel expression constructs. d. Evaluation of the response of PRE2-Luc in similar experiments to c. Error bars represent the s.e.m in b, c and d (n = 6 for each data point).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4221282&req=5

pone-0112247-g002: POU4F3-mediated activation of PRE1, PRE2 and a 4.2 kb Nr2f2 5′ flanking sequence.a. Schematic diagram of reporter constructs used in luciferase reporter assays in ND7 cells. POU4F3 recognition element (PRE) 1 and 2 are shown in an Nr2f2 5′ flanking region fragment (grey box) cloned upstream of a luciferase reporter gene (LUC). The location of this fragment relative to the Nr2f2 transcriptional start site is shown. b. Evaluation of the response of 4.2 kb-Nr2f2-Luc to increasing levels of POU4F3 or dreidel mutant POU4F3 (Ddl) expression construct. The luciferase activity of the reporter is normalised to its response to the empty expression vector and results are expressed relative to this. c. Response of the PRE1-Luc reporter construct in co-transfection experiments with POU4F3 and dreidel expression constructs. d. Evaluation of the response of PRE2-Luc in similar experiments to c. Error bars represent the s.e.m in b, c and d (n = 6 for each data point).
Mentions: Having demonstrated the ability of POU4F3 to bind to PRE1 and PRE2, we tested whether POU4F3 can regulate the Nr2f2 5′ flanking region that contains these sites. A luciferase reporter construct containing 4.2 kb of the Nr2f2 5′ flanking region (4.2 kb-Nr2f2-Luc, Figure 2a) was used in co-transfection studies in ND7 cells. This analysis demonstrated a dose-dependent increase in 4.2 kb-Nr2f2-Luc activity in response to increasing POU4F3 levels. Compared to basal activity, promoter activity was five times higher at the maximal amount of POU4F3 expression construct used (3 µg) and was not replicated with a non-DNA-binding POU4F3 mutant (dreidel), showing that this activation is dependent on POU4F3-specific DNA binding (Figure 2b).

Bottom Line: These sites were shown to be required for POU4F3 activation as their mutation leads to a reduction in the response of an Nr2f2 5' flanking region reporter construct to POU4F3.Immunocytochemistry was carried out in the developing and adult inner ear in order to investigate the relevance of this interaction in hearing.NR2F2 expression in the postnatal mouse organ of Corti was shown to be detectable in all sensory epithelia examined and characterised.

View Article: PubMed Central - PubMed

Affiliation: UCL Ear Institute, University College London, London, United Kingdom.

ABSTRACT
Hair cells are the mechanotransducing cells of the inner ear that are essential for hearing and balance. POU4F3--a POU-domain transcription factor selectively expressed by these cells--has been shown to be essential for hair cell differentiation and survival in mice and its mutation in humans underlies late-onset progressive hearing loss (DFNA15). The downstream targets of POU4F3 are required for hair cell differentiation and survival. We aimed to identify such targets in order to elucidate the molecular pathways involved in hair cell production and maintenance. The orphan thyroid nuclear receptor Nr2f2 was identified as a POU4F3 target using a subtractive hybridization strategy and EMSA analysis showed that POU4F3 binds to two sites in the Nr2f2 5' flanking region. These sites were shown to be required for POU4F3 activation as their mutation leads to a reduction in the response of an Nr2f2 5' flanking region reporter construct to POU4F3. Immunocytochemistry was carried out in the developing and adult inner ear in order to investigate the relevance of this interaction in hearing. NR2F2 expression in the postnatal mouse organ of Corti was shown to be detectable in all sensory epithelia examined and characterised. These data demonstrate that Nr2f2 is a direct target of POU4F3 in vitro and that this regulatory relationship may be relevant to hair cell development and survival.

Show MeSH
Related in: MedlinePlus