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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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Recruitment of Pol II to the promoter of the luciferase transgene in HELN-Fα/β cells. Kinetic ChIP experiments were performed using anti-Pol II antibody. Chromatin samples were obtained and data processed as described in Figure 4. The corresponding amplitudes of variation are: in A (HELN-Fα) Imin = 1.5, Imax = 3.1; in B (HELNβ) Imin = 2.5 Imax = 3.0.
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Figure 5: Recruitment of Pol II to the promoter of the luciferase transgene in HELN-Fα/β cells. Kinetic ChIP experiments were performed using anti-Pol II antibody. Chromatin samples were obtained and data processed as described in Figure 4. The corresponding amplitudes of variation are: in A (HELN-Fα) Imin = 1.5, Imax = 3.1; in B (HELNβ) Imin = 2.5 Imax = 3.0.

Mentions: The ultimate aim of these experiments was to analyse the respective characteristics of ERα and ERβ recruitment on the estrogen inducible stably transfected luciferase transgene within an identical cell context. The time scale that has already been studied with great details in the literature concerned the recruitment of ERα on the pS2 promoter during the first 180 minutes of stimulation [19]. In our study, we have chosen to focus on events that operate on a longer time scale (0-8 hr) on the luciferase transgene promoter. Transcription of cells was first reset by an α-amanitin treatment in order to synchronize and maximize the signal obtained after E2 stimulation. In fact, the effect of α-amanitin treatment is probably more important during the first two hours of estradiol stimulation, and has been used for the detailed analysis of cyclical recruitment of ERα in MCF-7 cells during this time scale [19]. After two hours of α-amanitin treatment, cells were treated with 10 nM of E2 during 8 hours, and cross-linked at indicated times (see methods and figure legend). One difficulty that was encountered was the strict reproducibility of the range of the % input scale when two independent kinetics of recruitment were compared, even though curves shapes were comparable. This can reveal subtle differences in the cell population which may occur among cell cultures (cell passages, cell density...). One representative kinetic ChIP experiment from two independent IPs performed on the same chromatin is shown (Figure 4 and 5). The range of variation of the maximum average value of one curve for all kinetics is indicated (see figure legend).


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)

Recruitment of Pol II to the promoter of the luciferase transgene in HELN-Fα/β cells. Kinetic ChIP experiments were performed using anti-Pol II antibody. Chromatin samples were obtained and data processed as described in Figure 4. The corresponding amplitudes of variation are: in A (HELN-Fα) Imin = 1.5, Imax = 3.1; in B (HELNβ) Imin = 2.5 Imax = 3.0.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Recruitment of Pol II to the promoter of the luciferase transgene in HELN-Fα/β cells. Kinetic ChIP experiments were performed using anti-Pol II antibody. Chromatin samples were obtained and data processed as described in Figure 4. The corresponding amplitudes of variation are: in A (HELN-Fα) Imin = 1.5, Imax = 3.1; in B (HELNβ) Imin = 2.5 Imax = 3.0.
Mentions: The ultimate aim of these experiments was to analyse the respective characteristics of ERα and ERβ recruitment on the estrogen inducible stably transfected luciferase transgene within an identical cell context. The time scale that has already been studied with great details in the literature concerned the recruitment of ERα on the pS2 promoter during the first 180 minutes of stimulation [19]. In our study, we have chosen to focus on events that operate on a longer time scale (0-8 hr) on the luciferase transgene promoter. Transcription of cells was first reset by an α-amanitin treatment in order to synchronize and maximize the signal obtained after E2 stimulation. In fact, the effect of α-amanitin treatment is probably more important during the first two hours of estradiol stimulation, and has been used for the detailed analysis of cyclical recruitment of ERα in MCF-7 cells during this time scale [19]. After two hours of α-amanitin treatment, cells were treated with 10 nM of E2 during 8 hours, and cross-linked at indicated times (see methods and figure legend). One difficulty that was encountered was the strict reproducibility of the range of the % input scale when two independent kinetics of recruitment were compared, even though curves shapes were comparable. This can reveal subtle differences in the cell population which may occur among cell cultures (cell passages, cell density...). One representative kinetic ChIP experiment from two independent IPs performed on the same chromatin is shown (Figure 4 and 5). The range of variation of the maximum average value of one curve for all kinetics is indicated (see figure legend).

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