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


CONCLUSIONS
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.
Badia
Eric
E
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
Escande
Aurélie
A
Balaguer
Patrick
P
Métivier
Raphaël
R
Cavailles
Vincent
V
D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D000939:Epitopes / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D047629:Estrogen Receptor beta / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D002843:Chromatin; D000939:Epitopes; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D008156:Luciferases