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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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Structure, expression and binding properties of the FLAG-ERα/β proteins. A) Representation of FLAG-ERα/β proteins showing the amino acid sequence length of ERα and ERβ, as well as their different domains, and the position of the FLAG tag. B) Western blot using the M2 anti-FLAG antibody to probe the relative amounts of FLAG-ERα or FLAG-ERβ present in 40 μg of total protein extracts prepared from HELN-Fα or HELN-Fβ stable cell lines cultured in 3% DCC (in absence of phenol red). C) Scatchard plot analysis of specific binding of [3H]-E2 to FLAG-ERα or FLAG-ERβ during 6 hours in a whole cell assay. Dissociation constant (Kd) is indicated.
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Figure 1: Structure, expression and binding properties of the FLAG-ERα/β proteins. A) Representation of FLAG-ERα/β proteins showing the amino acid sequence length of ERα and ERβ, as well as their different domains, and the position of the FLAG tag. B) Western blot using the M2 anti-FLAG antibody to probe the relative amounts of FLAG-ERα or FLAG-ERβ present in 40 μg of total protein extracts prepared from HELN-Fα or HELN-Fβ stable cell lines cultured in 3% DCC (in absence of phenol red). C) Scatchard plot analysis of specific binding of [3H]-E2 to FLAG-ERα or FLAG-ERβ during 6 hours in a whole cell assay. Dissociation constant (Kd) is indicated.

Mentions: The 3× FLAG epitope (FLAG) fused to the N-terminus of the receptors (Figure 1A) was used to analyse their expression by Western blotting in the different clones of HELN-Fα and HELN-Fβ obtained. Two cell lines that displayed comparable levels of FLAG-ERα and FLAG-ERβ (Figure 1B) were selected and then used for all following experiments. FLAG-ERα and FLAG-ERβ receptor protein levels, as well as their respective affinities for estradiol, were more precisely determined using 3H-estradiol in saturation experiments (Figure 1C). Scatchard analysis performed in whole cell assays, led to a protein determination of 50 fmoles/mg and 159 fmol/mg and a dissociation constant of 0.03 nM and 0.27 nM for FLAG-ERα and FLAG-ERβ respectively. These dissociation constant values were similar to those previously found for untagged receptors [27]. Indeed, in HELN-ERα and HELN-ERβ cell lines, Kd were 0.04 and 0.11 nM respectively thus indicating that the FLAG tag do not interfere with estradiol binding. In the parental HELN cell line, ERs (α or β) were not detected by binding experiments [27].


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)

Structure, expression and binding properties of the FLAG-ERα/β proteins. A) Representation of FLAG-ERα/β proteins showing the amino acid sequence length of ERα and ERβ, as well as their different domains, and the position of the FLAG tag. B) Western blot using the M2 anti-FLAG antibody to probe the relative amounts of FLAG-ERα or FLAG-ERβ present in 40 μg of total protein extracts prepared from HELN-Fα or HELN-Fβ stable cell lines cultured in 3% DCC (in absence of phenol red). C) Scatchard plot analysis of specific binding of [3H]-E2 to FLAG-ERα or FLAG-ERβ during 6 hours in a whole cell assay. Dissociation constant (Kd) is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Structure, expression and binding properties of the FLAG-ERα/β proteins. A) Representation of FLAG-ERα/β proteins showing the amino acid sequence length of ERα and ERβ, as well as their different domains, and the position of the FLAG tag. B) Western blot using the M2 anti-FLAG antibody to probe the relative amounts of FLAG-ERα or FLAG-ERβ present in 40 μg of total protein extracts prepared from HELN-Fα or HELN-Fβ stable cell lines cultured in 3% DCC (in absence of phenol red). C) Scatchard plot analysis of specific binding of [3H]-E2 to FLAG-ERα or FLAG-ERβ during 6 hours in a whole cell assay. Dissociation constant (Kd) is indicated.
Mentions: The 3× FLAG epitope (FLAG) fused to the N-terminus of the receptors (Figure 1A) was used to analyse their expression by Western blotting in the different clones of HELN-Fα and HELN-Fβ obtained. Two cell lines that displayed comparable levels of FLAG-ERα and FLAG-ERβ (Figure 1B) were selected and then used for all following experiments. FLAG-ERα and FLAG-ERβ receptor protein levels, as well as their respective affinities for estradiol, were more precisely determined using 3H-estradiol in saturation experiments (Figure 1C). Scatchard analysis performed in whole cell assays, led to a protein determination of 50 fmoles/mg and 159 fmol/mg and a dissociation constant of 0.03 nM and 0.27 nM for FLAG-ERα and FLAG-ERβ respectively. These dissociation constant values were similar to those previously found for untagged receptors [27]. Indeed, in HELN-ERα and HELN-ERβ cell lines, Kd were 0.04 and 0.11 nM respectively thus indicating that the FLAG tag do not interfere with estradiol binding. In the parental HELN cell line, ERs (α or β) were not detected by binding experiments [27].

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