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AP-1-mediated expression of brain-specific class IVa β-tubulin in P19 embryonal carcinoma cells.

Maruyama Y, Arahara K, Kinoshita E, Arai K - J. Vet. Med. Sci. (2014)

Bottom Line: Transient transfection assays employing a reporter construct found that ATRA-mediated regulatory region of the TUBB4a gene lay in the region from -83 nt to +137 nt relative to the +1 transcription start site.Further immunoblot study revealed an ATRA-mediated increase in JunD protein, while a significant amount of ATF2 protein was constantly produced.These results suggest that differentiation-mediated activation of JunD results in enhanced TUBB4a expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan.

ABSTRACT
Expression of brain-specific phenotypes increased in all trans retinoic acid (ATRA)-induced neural differentiation of mouse P19 embryonal carcinoma cells. Among these phenotypes, expression of class IVa β-tubulin isotype (TUBB4a) was particularly enhanced in neural differentiation. Transient transfection assays employing a reporter construct found that ATRA-mediated regulatory region of the TUBB4a gene lay in the region from -83 nt to +137 nt relative to the +1 transcription start site. Site-directed mutagenesis in the AP-1 binding site at -29/-17 suggested that the AP-1 binding site was a critical region for ATRA-mediated TUBB4a expression. Chromatin immunoprecipitation experiments suggested participation of JunD and activating transcription factor-2 (ATF2) in TUBB4a expression. Additionally, exogenous induction of the dominant-negative (dn) type of JunD canceled ATRA-induced upregulation of TUBB4a, and the dn type of ATF2 suppressed even the basal activity. Further immunoblot study revealed an ATRA-mediated increase in JunD protein, while a significant amount of ATF2 protein was constantly produced. These results suggest that differentiation-mediated activation of JunD results in enhanced TUBB4a expression.

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A. Effect of the dominant-negative forms of c-Jun, JunD c-Fos, CREB1 or ATF2 on thestimulation of promoter activity for mTUBB4aS mediated by neuronaldifferentiation. Control (C) and ATRA-treated (D) P19 cells transfected withpmTUBB4aS-luc,pRL-tk and one of the expression vectors or theempty vector pcDNA3. Luciferase activity was measured and normalized against theRenilla luciferase activity. The values shown are mean ± SD ofthree representative and independent experiments. Asterisks indicate a significantdifference (P<0.05) from undifferentiated cells. B. Western blotof cellular protein extracts from control (C) and differentiated (D) P19 cells usingan antibody to JunD or ATF2. mw: molecular weight.
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fig_004: A. Effect of the dominant-negative forms of c-Jun, JunD c-Fos, CREB1 or ATF2 on thestimulation of promoter activity for mTUBB4aS mediated by neuronaldifferentiation. Control (C) and ATRA-treated (D) P19 cells transfected withpmTUBB4aS-luc,pRL-tk and one of the expression vectors or theempty vector pcDNA3. Luciferase activity was measured and normalized against theRenilla luciferase activity. The values shown are mean ± SD ofthree representative and independent experiments. Asterisks indicate a significantdifference (P<0.05) from undifferentiated cells. B. Western blotof cellular protein extracts from control (C) and differentiated (D) P19 cells usingan antibody to JunD or ATF2. mw: molecular weight.

Mentions: Effect of forced expression of transcription factors in the AP-1 family on theluciferase activity of TUBB4 gene: The ChIP assay indicated that JunD and ATF2bound to the regulatory region of the mTUBB4a gene. To further confirmthis, forced expression of AP-1 family genes in P19 cells was performed. As a result,co-transfection of dominant-negative JunD or ATF2 abrogated the effect of ATRA on thetranscriptional activity of mTUBB4aS (Fig. 4AFig. 4.


AP-1-mediated expression of brain-specific class IVa β-tubulin in P19 embryonal carcinoma cells.

Maruyama Y, Arahara K, Kinoshita E, Arai K - J. Vet. Med. Sci. (2014)

A. Effect of the dominant-negative forms of c-Jun, JunD c-Fos, CREB1 or ATF2 on thestimulation of promoter activity for mTUBB4aS mediated by neuronaldifferentiation. Control (C) and ATRA-treated (D) P19 cells transfected withpmTUBB4aS-luc,pRL-tk and one of the expression vectors or theempty vector pcDNA3. Luciferase activity was measured and normalized against theRenilla luciferase activity. The values shown are mean ± SD ofthree representative and independent experiments. Asterisks indicate a significantdifference (P<0.05) from undifferentiated cells. B. Western blotof cellular protein extracts from control (C) and differentiated (D) P19 cells usingan antibody to JunD or ATF2. mw: molecular weight.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300376&req=5

fig_004: A. Effect of the dominant-negative forms of c-Jun, JunD c-Fos, CREB1 or ATF2 on thestimulation of promoter activity for mTUBB4aS mediated by neuronaldifferentiation. Control (C) and ATRA-treated (D) P19 cells transfected withpmTUBB4aS-luc,pRL-tk and one of the expression vectors or theempty vector pcDNA3. Luciferase activity was measured and normalized against theRenilla luciferase activity. The values shown are mean ± SD ofthree representative and independent experiments. Asterisks indicate a significantdifference (P<0.05) from undifferentiated cells. B. Western blotof cellular protein extracts from control (C) and differentiated (D) P19 cells usingan antibody to JunD or ATF2. mw: molecular weight.
Mentions: Effect of forced expression of transcription factors in the AP-1 family on theluciferase activity of TUBB4 gene: The ChIP assay indicated that JunD and ATF2bound to the regulatory region of the mTUBB4a gene. To further confirmthis, forced expression of AP-1 family genes in P19 cells was performed. As a result,co-transfection of dominant-negative JunD or ATF2 abrogated the effect of ATRA on thetranscriptional activity of mTUBB4aS (Fig. 4AFig. 4.

Bottom Line: Transient transfection assays employing a reporter construct found that ATRA-mediated regulatory region of the TUBB4a gene lay in the region from -83 nt to +137 nt relative to the +1 transcription start site.Further immunoblot study revealed an ATRA-mediated increase in JunD protein, while a significant amount of ATF2 protein was constantly produced.These results suggest that differentiation-mediated activation of JunD results in enhanced TUBB4a expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Tissue Physiology, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan.

ABSTRACT
Expression of brain-specific phenotypes increased in all trans retinoic acid (ATRA)-induced neural differentiation of mouse P19 embryonal carcinoma cells. Among these phenotypes, expression of class IVa β-tubulin isotype (TUBB4a) was particularly enhanced in neural differentiation. Transient transfection assays employing a reporter construct found that ATRA-mediated regulatory region of the TUBB4a gene lay in the region from -83 nt to +137 nt relative to the +1 transcription start site. Site-directed mutagenesis in the AP-1 binding site at -29/-17 suggested that the AP-1 binding site was a critical region for ATRA-mediated TUBB4a expression. Chromatin immunoprecipitation experiments suggested participation of JunD and activating transcription factor-2 (ATF2) in TUBB4a expression. Additionally, exogenous induction of the dominant-negative (dn) type of JunD canceled ATRA-induced upregulation of TUBB4a, and the dn type of ATF2 suppressed even the basal activity. Further immunoblot study revealed an ATRA-mediated increase in JunD protein, while a significant amount of ATF2 protein was constantly produced. These results suggest that differentiation-mediated activation of JunD results in enhanced TUBB4a expression.

Show MeSH
Related in: MedlinePlus