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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. Presumptive transcription factor binding sites of the promoter region and firstintron of mTUBB4aM and TUBB4aS. A. The sequencecontained within nucleotides −360 and + 534 nt of the mTUBB4aM genewas analyzed using a web-based search engine. Two putative AP-1 sites (closed box) andfive Sp1 sites (closed oval) are indicated, and the first exon of the gene is boxed;an asterisk indicates a transcription start site. Two arrows indicate primer sites ofthe amplified region for the ChIP assay. The mutant in the AP-1binding site (−29/−17)within the promoter region of mTUBB4aS is indicated as AP-1m. Thesequence of the AP-1 site is CATCAGACGCCAC, while that of AP-1m is CATCAGACGCttt;lowercase letters indicates the mutation site. B. EMSA was performed using DIG-labeledwild and AP-1-mutated probes. Nuclear protein extracted from undifferentiated P19cells and cells undergoing neural differentiation: lanes 1 and 5, nuclear protein fromcontrol cells; lanes 2–4 and 6, nuclear protein from neurodifferentiated P19 cells.Competition experiments were performed with a 250-fold excess of an unlabeledconsensus AP-1 probe (lane 3) and the mutated probe (lane 4). EMSA with a DIG-labeledmutated probe is shown in lanes 5 and 6. C. Control (C) and ATRA-treated P19 cells (D)were transfected with 1 µg of the pmTUBB4aS-luc orits mutants and 100 ng of pRL-tkfor 5 hr. The medium was replaced, incubation was performed overnight, the cell layerswere harvested, and then the luciferase activity was measured and normalized toRenilla luciferase activity. The values are means ± SD and arerepresentative of three independent experiments. D. Control (C) and ATRA-treated P19cells (D) were used for ChIP-qPCR. The antibodies to c-Jun, JunD, c-Fos, CREB1 andATF2 used for immunoprecipitation are indicated. Anti-histone H3 antibody (H3) wasused for the positive control. Data are shown as means ± SD of three independentexperiments.
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fig_003: A. Presumptive transcription factor binding sites of the promoter region and firstintron of mTUBB4aM and TUBB4aS. A. The sequencecontained within nucleotides −360 and + 534 nt of the mTUBB4aM genewas analyzed using a web-based search engine. Two putative AP-1 sites (closed box) andfive Sp1 sites (closed oval) are indicated, and the first exon of the gene is boxed;an asterisk indicates a transcription start site. Two arrows indicate primer sites ofthe amplified region for the ChIP assay. The mutant in the AP-1binding site (−29/−17)within the promoter region of mTUBB4aS is indicated as AP-1m. Thesequence of the AP-1 site is CATCAGACGCCAC, while that of AP-1m is CATCAGACGCttt;lowercase letters indicates the mutation site. B. EMSA was performed using DIG-labeledwild and AP-1-mutated probes. Nuclear protein extracted from undifferentiated P19cells and cells undergoing neural differentiation: lanes 1 and 5, nuclear protein fromcontrol cells; lanes 2–4 and 6, nuclear protein from neurodifferentiated P19 cells.Competition experiments were performed with a 250-fold excess of an unlabeledconsensus AP-1 probe (lane 3) and the mutated probe (lane 4). EMSA with a DIG-labeledmutated probe is shown in lanes 5 and 6. C. Control (C) and ATRA-treated P19 cells (D)were transfected with 1 µg of the pmTUBB4aS-luc orits mutants and 100 ng of pRL-tkfor 5 hr. The medium was replaced, incubation was performed overnight, the cell layerswere harvested, and then the luciferase activity was measured and normalized toRenilla luciferase activity. The values are means ± SD and arerepresentative of three independent experiments. D. Control (C) and ATRA-treated P19cells (D) were used for ChIP-qPCR. The antibodies to c-Jun, JunD, c-Fos, CREB1 andATF2 used for immunoprecipitation are indicated. Anti-histone H3 antibody (H3) wasused for the positive control. Data are shown as means ± SD of three independentexperiments.

Mentions: Schematic representation of the promoter region of the mTUBB4a gene.Three different lengths of PCR products were amplified and subcloned into pGL3-basicvector. The closed box indicates the first exon. P19 cells were pretreated to formneurospheres as described above, and then the neurospheres were dispersed and culturedon gelatin-coated dishes. After overnight culture, control (C) and ATRA-treated P19cells (D) were transfected with pGL3-basic vector subcloned with mTUBB4aL(−1145/+504), mTUBB4aM (−360/+534) and mTUBB4aS(−83/+137), respectively, and then cultured overnight. At the end ofcultivation, the luciferase activity in the supernatant of cell lysate was analyzed.The values shown are means ± SD of three independent experiments. Asterisks indicate asignificant difference (P<0.05) from undifferentiated controlcells (C).


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. Presumptive transcription factor binding sites of the promoter region and firstintron of mTUBB4aM and TUBB4aS. A. The sequencecontained within nucleotides −360 and + 534 nt of the mTUBB4aM genewas analyzed using a web-based search engine. Two putative AP-1 sites (closed box) andfive Sp1 sites (closed oval) are indicated, and the first exon of the gene is boxed;an asterisk indicates a transcription start site. Two arrows indicate primer sites ofthe amplified region for the ChIP assay. The mutant in the AP-1binding site (−29/−17)within the promoter region of mTUBB4aS is indicated as AP-1m. Thesequence of the AP-1 site is CATCAGACGCCAC, while that of AP-1m is CATCAGACGCttt;lowercase letters indicates the mutation site. B. EMSA was performed using DIG-labeledwild and AP-1-mutated probes. Nuclear protein extracted from undifferentiated P19cells and cells undergoing neural differentiation: lanes 1 and 5, nuclear protein fromcontrol cells; lanes 2–4 and 6, nuclear protein from neurodifferentiated P19 cells.Competition experiments were performed with a 250-fold excess of an unlabeledconsensus AP-1 probe (lane 3) and the mutated probe (lane 4). EMSA with a DIG-labeledmutated probe is shown in lanes 5 and 6. C. Control (C) and ATRA-treated P19 cells (D)were transfected with 1 µg of the pmTUBB4aS-luc orits mutants and 100 ng of pRL-tkfor 5 hr. The medium was replaced, incubation was performed overnight, the cell layerswere harvested, and then the luciferase activity was measured and normalized toRenilla luciferase activity. The values are means ± SD and arerepresentative of three independent experiments. D. Control (C) and ATRA-treated P19cells (D) were used for ChIP-qPCR. The antibodies to c-Jun, JunD, c-Fos, CREB1 andATF2 used for immunoprecipitation are indicated. Anti-histone H3 antibody (H3) wasused for the positive control. Data are shown as means ± SD of three independentexperiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig_003: A. Presumptive transcription factor binding sites of the promoter region and firstintron of mTUBB4aM and TUBB4aS. A. The sequencecontained within nucleotides −360 and + 534 nt of the mTUBB4aM genewas analyzed using a web-based search engine. Two putative AP-1 sites (closed box) andfive Sp1 sites (closed oval) are indicated, and the first exon of the gene is boxed;an asterisk indicates a transcription start site. Two arrows indicate primer sites ofthe amplified region for the ChIP assay. The mutant in the AP-1binding site (−29/−17)within the promoter region of mTUBB4aS is indicated as AP-1m. Thesequence of the AP-1 site is CATCAGACGCCAC, while that of AP-1m is CATCAGACGCttt;lowercase letters indicates the mutation site. B. EMSA was performed using DIG-labeledwild and AP-1-mutated probes. Nuclear protein extracted from undifferentiated P19cells and cells undergoing neural differentiation: lanes 1 and 5, nuclear protein fromcontrol cells; lanes 2–4 and 6, nuclear protein from neurodifferentiated P19 cells.Competition experiments were performed with a 250-fold excess of an unlabeledconsensus AP-1 probe (lane 3) and the mutated probe (lane 4). EMSA with a DIG-labeledmutated probe is shown in lanes 5 and 6. C. Control (C) and ATRA-treated P19 cells (D)were transfected with 1 µg of the pmTUBB4aS-luc orits mutants and 100 ng of pRL-tkfor 5 hr. The medium was replaced, incubation was performed overnight, the cell layerswere harvested, and then the luciferase activity was measured and normalized toRenilla luciferase activity. The values are means ± SD and arerepresentative of three independent experiments. D. Control (C) and ATRA-treated P19cells (D) were used for ChIP-qPCR. The antibodies to c-Jun, JunD, c-Fos, CREB1 andATF2 used for immunoprecipitation are indicated. Anti-histone H3 antibody (H3) wasused for the positive control. Data are shown as means ± SD of three independentexperiments.
Mentions: Schematic representation of the promoter region of the mTUBB4a gene.Three different lengths of PCR products were amplified and subcloned into pGL3-basicvector. The closed box indicates the first exon. P19 cells were pretreated to formneurospheres as described above, and then the neurospheres were dispersed and culturedon gelatin-coated dishes. After overnight culture, control (C) and ATRA-treated P19cells (D) were transfected with pGL3-basic vector subcloned with mTUBB4aL(−1145/+504), mTUBB4aM (−360/+534) and mTUBB4aS(−83/+137), respectively, and then cultured overnight. At the end ofcultivation, the luciferase activity in the supernatant of cell lysate was analyzed.The values shown are means ± SD of three independent experiments. Asterisks indicate asignificant difference (P<0.05) from undifferentiated controlcells (C).

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