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New stably transfected bioluminescent cells expressing FLAG epitope-tagged estrogen receptors to study their chromatin recruitment.

Badia E, Escande A, Balaguer P, Métivier R, Cavailles V - BMC Biotechnol. (2009)

Bottom Line: HELN-Falpha and HELN-Fbeta cell lines were found to express comparable levels of their corresponding tagged receptors with a Kd for estradiol binding of 0.03 and 0.27 nM respectively.FLAG-ERalpha and FLAG-ERbeta were found to exhibit similar transcriptional activity, as indicated by a kinetic evaluation of the transcriptional activation of the luciferase gene during 10 hrs of treatment with estradiol.However, the relative importance of recruitment between 1 hr and 4 hr was found to be different in HeLa cell line expressing exogenous tagged ERalpha and in MCF-7 cell line expressing endogenous ER.

View Article: PubMed Central - HTML - PubMed

Affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, F-34298; INSERM, U896, F-34298; Université Montpellier1, F-34298; CRLC Val d'Aurelle Paul Lamarque, Montpellier, F-34298, France. e.badia@valdorel.fnclcc.fr

ABSTRACT

Background: Biological actions of estrogens are mediated by the two specific estrogen receptors ERalpha and ERbeta. However, due to the absence of adequate cellular models, their respective transcriptional activities are still poorly understood. For instance, the evaluation of such differing properties on the transcription of responsive genes using ChIP experiments was hindered by the deficiency of cells exhibiting the same genotypic background and properties but expressing only one of the ERs. We describe here the generation of such cells, using an estrogen receptor negative HELN cell line that was derived from HeLa cells stably transfected with an ERE-driven luciferase plasmid. These HELN-Falpha and HELN-Fbeta cell lines stably express either the alpha or beta (full length) estrogen receptor tagged with the FLAG epitope. The use of antibodies directed against the FLAG epitope allowed a direct comparative evaluation of the respective actions of both ERs using ChIP.

Results: HELN-Falpha and HELN-Fbeta cell lines were found to express comparable levels of their corresponding tagged receptors with a Kd for estradiol binding of 0.03 and 0.27 nM respectively. The presence of a stably transfected ERE-driven luciferase plasmid in these cells allowed the direct evaluation of the transcriptional activity of both tagged receptors, using natural or synthetic estrogens. FLAG-ERalpha and FLAG-ERbeta were found to exhibit similar transcriptional activity, as indicated by a kinetic evaluation of the transcriptional activation of the luciferase gene during 10 hrs of treatment with estradiol. The validity of these model cells was further confirmed by the predictable transcriptional regulations measured upon treatments with ERalpha or ERbeta specific ligands. The similar immunoprecipitation efficiency of both tagged receptors by an anti-FLAG antibody allowed the assessment of their kinetic recruitment on the synthetic luciferase promoter (containing an estrogen response element) by ChIP assays during 8 hours. A biphasic curve was obtained for both FLAG-ERalpha and FLAG-ERbeta, with a peak occurring either at 2 hr or at 1 hr, respectively, and a second one following 4 hr of E2 stimulation in both cases. In MCF-7 cells, the recruitment of ERalpha also exhibited a biphasic behaviour; with the second peak however not so important than in the HeLa cell lines.

Conclusion: In HELN derived cell lines, no fundamental differences between kinetics were observed during 8 hours for FLAG-ERalpha and FLAG-ERbeta, as well as for polymerase II recruitment. However, the relative importance of recruitment between 1 hr and 4 hr was found to be different in HeLa cell line expressing exogenous tagged ERalpha and in MCF-7 cell line expressing endogenous ER.

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Kinetic analysis of luciferase induced expression in HELN-Fα/β cells and effect of selective agonist ligands on transcriptional activity. A) Kinetic analysis of luciferase expression of HELN-Fα and HELN-Fβ stable cell lines stimulated by 10 nM E2 during 10 hours. Luciferase activity was then recorded every hour and expressed as the percentage of maximal activity. HELN-Fα or HELN-Fβ stable cell lines were stimulated 16 hr with either E2 (B), DPN (C) PPT (D) at indicated concentrations. Maximal activities (100%) correspond to the activity obtained with a 10 nM E2 stimulation. Values are mean ± S.D. from quadruplicate experiments. In A, B, C, D, filled circle were for HELN-Fα and empty circles for HELN-Fβ.
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Figure 2: Kinetic analysis of luciferase induced expression in HELN-Fα/β cells and effect of selective agonist ligands on transcriptional activity. A) Kinetic analysis of luciferase expression of HELN-Fα and HELN-Fβ stable cell lines stimulated by 10 nM E2 during 10 hours. Luciferase activity was then recorded every hour and expressed as the percentage of maximal activity. HELN-Fα or HELN-Fβ stable cell lines were stimulated 16 hr with either E2 (B), DPN (C) PPT (D) at indicated concentrations. Maximal activities (100%) correspond to the activity obtained with a 10 nM E2 stimulation. Values are mean ± S.D. from quadruplicate experiments. In A, B, C, D, filled circle were for HELN-Fα and empty circles for HELN-Fβ.

Mentions: A comparative study of the transactivation properties of each of the tagged receptors was next undertaken by analysing the luciferase activity of HELN-Fα and HELN-Fβ cell lines upon estradiol treatments. Firstly, we performed a kinetic analysis of ERE-driven luciferase gene induction during prolonged estradiol stimulation (10 hr), which is an agonist ligand for both receptors. As shown in Figure 2A, a significant and similar induction of luciferase activity was detected in both cell lines containing the respective tagged version of each receptor. Luciferase activities expressed per mg of protein also confirm the homogeneity of the hormonal response obtained in these two cell lines (data not shown).


New stably transfected bioluminescent cells expressing FLAG epitope-tagged estrogen receptors to study their chromatin recruitment.

Badia E, Escande A, Balaguer P, Métivier R, Cavailles V - BMC Biotechnol. (2009)

Kinetic analysis of luciferase induced expression in HELN-Fα/β cells and effect of selective agonist ligands on transcriptional activity. A) Kinetic analysis of luciferase expression of HELN-Fα and HELN-Fβ stable cell lines stimulated by 10 nM E2 during 10 hours. Luciferase activity was then recorded every hour and expressed as the percentage of maximal activity. HELN-Fα or HELN-Fβ stable cell lines were stimulated 16 hr with either E2 (B), DPN (C) PPT (D) at indicated concentrations. Maximal activities (100%) correspond to the activity obtained with a 10 nM E2 stimulation. Values are mean ± S.D. from quadruplicate experiments. In A, B, C, D, filled circle were for HELN-Fα and empty circles for HELN-Fβ.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2749030&req=5

Figure 2: Kinetic analysis of luciferase induced expression in HELN-Fα/β cells and effect of selective agonist ligands on transcriptional activity. A) Kinetic analysis of luciferase expression of HELN-Fα and HELN-Fβ stable cell lines stimulated by 10 nM E2 during 10 hours. Luciferase activity was then recorded every hour and expressed as the percentage of maximal activity. HELN-Fα or HELN-Fβ stable cell lines were stimulated 16 hr with either E2 (B), DPN (C) PPT (D) at indicated concentrations. Maximal activities (100%) correspond to the activity obtained with a 10 nM E2 stimulation. Values are mean ± S.D. from quadruplicate experiments. In A, B, C, D, filled circle were for HELN-Fα and empty circles for HELN-Fβ.
Mentions: A comparative study of the transactivation properties of each of the tagged receptors was next undertaken by analysing the luciferase activity of HELN-Fα and HELN-Fβ cell lines upon estradiol treatments. Firstly, we performed a kinetic analysis of ERE-driven luciferase gene induction during prolonged estradiol stimulation (10 hr), which is an agonist ligand for both receptors. As shown in Figure 2A, a significant and similar induction of luciferase activity was detected in both cell lines containing the respective tagged version of each receptor. Luciferase activities expressed per mg of protein also confirm the homogeneity of the hormonal response obtained in these two cell lines (data not shown).

Bottom Line: HELN-Falpha and HELN-Fbeta cell lines were found to express comparable levels of their corresponding tagged receptors with a Kd for estradiol binding of 0.03 and 0.27 nM respectively.FLAG-ERalpha and FLAG-ERbeta were found to exhibit similar transcriptional activity, as indicated by a kinetic evaluation of the transcriptional activation of the luciferase gene during 10 hrs of treatment with estradiol.However, the relative importance of recruitment between 1 hr and 4 hr was found to be different in HeLa cell line expressing exogenous tagged ERalpha and in MCF-7 cell line expressing endogenous ER.

View Article: PubMed Central - HTML - PubMed

Affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, F-34298; INSERM, U896, F-34298; Université Montpellier1, F-34298; CRLC Val d'Aurelle Paul Lamarque, Montpellier, F-34298, France. e.badia@valdorel.fnclcc.fr

ABSTRACT

Background: Biological actions of estrogens are mediated by the two specific estrogen receptors ERalpha and ERbeta. However, due to the absence of adequate cellular models, their respective transcriptional activities are still poorly understood. For instance, the evaluation of such differing properties on the transcription of responsive genes using ChIP experiments was hindered by the deficiency of cells exhibiting the same genotypic background and properties but expressing only one of the ERs. We describe here the generation of such cells, using an estrogen receptor negative HELN cell line that was derived from HeLa cells stably transfected with an ERE-driven luciferase plasmid. These HELN-Falpha and HELN-Fbeta cell lines stably express either the alpha or beta (full length) estrogen receptor tagged with the FLAG epitope. The use of antibodies directed against the FLAG epitope allowed a direct comparative evaluation of the respective actions of both ERs using ChIP.

Results: HELN-Falpha and HELN-Fbeta cell lines were found to express comparable levels of their corresponding tagged receptors with a Kd for estradiol binding of 0.03 and 0.27 nM respectively. The presence of a stably transfected ERE-driven luciferase plasmid in these cells allowed the direct evaluation of the transcriptional activity of both tagged receptors, using natural or synthetic estrogens. FLAG-ERalpha and FLAG-ERbeta were found to exhibit similar transcriptional activity, as indicated by a kinetic evaluation of the transcriptional activation of the luciferase gene during 10 hrs of treatment with estradiol. The validity of these model cells was further confirmed by the predictable transcriptional regulations measured upon treatments with ERalpha or ERbeta specific ligands. The similar immunoprecipitation efficiency of both tagged receptors by an anti-FLAG antibody allowed the assessment of their kinetic recruitment on the synthetic luciferase promoter (containing an estrogen response element) by ChIP assays during 8 hours. A biphasic curve was obtained for both FLAG-ERalpha and FLAG-ERbeta, with a peak occurring either at 2 hr or at 1 hr, respectively, and a second one following 4 hr of E2 stimulation in both cases. In MCF-7 cells, the recruitment of ERalpha also exhibited a biphasic behaviour; with the second peak however not so important than in the HeLa cell lines.

Conclusion: In HELN derived cell lines, no fundamental differences between kinetics were observed during 8 hours for FLAG-ERalpha and FLAG-ERbeta, as well as for polymerase II recruitment. However, the relative importance of recruitment between 1 hr and 4 hr was found to be different in HeLa cell line expressing exogenous tagged ERalpha and in MCF-7 cell line expressing endogenous ER.

Show MeSH
Related in: MedlinePlus