79 research outputs found
New stably transfected bioluminescent cells expressing FLAG epitope-tagged estrogen receptors to study their chromatin recruitment
International audienceBACKGROUND: 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
The AF-1-deficient estrogen receptor ERα46 isoform is frequently expressed in human breast tumors
Are 'omics of estrogen receptors defining potential targets for breast cancer treatment?
International audienc
Regulation of transcription : A dynamic integration of multi-leveled information.
National audienc
Defining specificity of transcription factor regulatory activities.
International audienceMammalian transcription factors (TFs) are often involved in differential cell-type- and context-specific transcriptional responses. Recent large-scale comparative studies of TF recruitment to the genome, and of chromatin structure and gene expression, have allowed a better understanding of the general rules that underlie the differential activities of a given TF. It has emerged that chromatin structure dictates the differential binding of a given TF to cell-type-specific cis-regulatory elements. The subsequent regulation of TF activity then ensures the functional activation of only the precise subset of all regulatory sites bound by the TF that are required to mediate appropriate gene expression. Ultimately, the organization of the genome within the nucleus, and crosstalk between different cis-regulatory regions involved in gene regulation, also participate in establishing a specific transcriptional program. In this Commentary, we discuss how the integration of these different and probably intimately linked regulatory mechanisms allow for TF cell-type- and context-specific modulation of gene expression
Marking time: The dynamic role of chromatin and covalent modification in transcription.
International audienceThe expression of genes subject to strict regulation can be a highly dynamic, cyclical process that sequentially achieves and then limits transcription. Kinetic investigations of the estrogen responsive pS2 (TFF1) promoter, to determine the occupancy of factors or the occurrence of covalent marks on chromatin, have provided the most comprehensive picture of the complexity of transcriptional cycling to date. Cycles are initiated by the assembly of intermediate transcription factors that in turn provoke conscription of the basal transcription machinery. These events then achieve activation of the polymerase II complex, which is subsequently followed by limitation of productivity through the action of repressive complexes. This latter phase resets the target promoter, through acting on chromatin structure, such that a subsequent cycle can be initiated. In consequence, transcription is dependent upon cis-acting elements (DNA and nucleosomes) that either interact with or are modified by trans-acting factors. Induced local structural changes to chromatin encompassing regulatory elements of gene promoters include alteration of the positional phasing of nucleosomes, substitution by variant histones, post-translational modification of nucleosomes, changes in the methylation of CpG dinucleotides and breaks in the sugar-phosphate backbone of DNA. A primary function of covalent modification of chromatin may be to drive a sequential progression of reversible interactions that achieve and regulate gene expression
COUP-TFI and estrogen receptor interplay in breast cancer : evidence for in vivo regulation of Cathepsin D
Poste
Synergism between a half-site and an imperfect estrogen-responsive element, and cooperation with COUP-TFI are required for estrogen receptor (ER) to achieve a maximal estrogen-stimulation of rainbow trout ER gene
International audienc
Function of N-Terminal Transactivation Domain of the Estrogen Receptor Requires a Potential α-Helical Structure and Is Negatively Regulated by the A Domain
International audienc
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