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. 2004 Sep 15;23(18):3653-66.
doi: 10.1038/sj.emboj.7600377. Epub 2004 Sep 2.

Transcriptional complexes engaged by apo-estrogen receptor-alpha isoforms have divergent outcomes

Raphaël Métivier  1 , Graziella Penot, Richard P Carmouche, Michael R Hübner, George Reid, Stefanie Denger, Dominique Manu, Heike Brand, Martin Kos, Vladimir Benes, Frank Gannon
Affiliations

Transcriptional complexes engaged by apo-estrogen receptor-alpha isoforms have divergent outcomes

Raphaël Métivier et al. EMBO J. .

Abstract

Unliganded (apo-) estrogen receptor alpha (ERalpha, NR3A1) is classically considered as transcriptionally unproductive. Reassessing this paradigm demonstrated that apo-human ERalpha (ERalpha66) and its N-terminally truncated isoform (ERalpha46) are both predominantly nuclear transcription factors that cycle on the endogenous estrogen-responsive pS2 gene promoter in vivo. Importantly, isoform-specific consequences occur in terms of poising the promoter for transcription, as evaluated by determining (i) the engagement of several cofactors and the resulting nucleosomal organization; and (ii) the CpG methylation state of the pS2 promoter. Although transcriptionally unproductive, cycling of apo-ERalpha66 prepares the promoter to respond to ligand, through sequentially targeting chromatin remodeling complexes and general transcription factors. Additionally, apo-ERalpha46 recruits corepressors, following engagement of cofactors identical to those recruited by apo-ERalpha66. Together, these data describe differential activities of ERalpha isoforms. Furthermore, they depict the maintenance of a promoter in a repressed state as a cyclical process that is intrinsically dependent on initial poising of the promoter.

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Figures

Figure 1
Figure 1
Functional properties of ERα isoforms. (A) Scheme illustrating ERα isoforms sequence and the location of the epitopes targeted by antibodies used. (B) Immunostaining of ERα isoforms with HC20. MCF-7 cells expressing both isoforms and MDA-MB231 cells expressing none or either isoforms (MDA∷ERα66 or MDA∷ERα46) were fixed after a 3 h treatment with 10−8 M estradiol (E2) or ethanol (EtOH) as vehicle control. Nuclei are visualized by Hoechst staining. (C) The expression of endogenous pS2 gene in the four cell lines was monitored by real-time PCR. After 72 h of culture in stripped media, cells were treated for 3 h with 10−8 M E2 or EtOH as vehicle control. pS2 mRNA levels were normalized against invariant GAPDH mRNA. (D) ChIP probed the recruitment of ERα, P-PolII and Ac-Hist to the pS2 promoter in unsynchronized cells sampled as in (C).
Figure 2
Figure 2
Evaluation of intercellular variations in MeCpG content of the pS2 promoter. (A) Schematic representation of the pS2 promoter, with significant cis-acting features and phased nucleosomes (Sewack and Hansen, 1997) highlighted. CG dinucleotides are indicated, with those within an HpaII site in gray. Regions amplified by PCR primers are also depicted. (B) Genomic DNA prepared from MDA-MB231, MCF-7 and MDA-MB231 cells stably expressing either ERα isoforms was digested by HpaII and then subject to PCR to amplify the described regions. (C) To quantify the methylation of given CpG, genomic DNA was chemically modified using bisulfite-mediated C to U transformation. The upper strand of the pS2 promoter was then specifically amplified by PCR, and the total pool of fragments was sequenced. Values are the mean±s.e.m. from data acquired in three separate experiments.
Figure 3
Figure 3
Cyclical permissiveness of the pS2 promoter for ERα46 and ERα66 bindings, with distinct functional consequences. (A) Kinetic ChIP experiments performed using H184 or HC20 antibodies. Cells were first synchronized by a 72 h deprivation of serum and then treated for 2 h with 2.5 μM α-amanitin. After washing and renewal of the media, chromatin was prepared on sampled cells with 5 min intervals. Amounts of immunoprecipitated pS2 promoter were quantified by real-time PCR. Values, expressed as the percent of inputs, are the mean of three separate experiments, and have an s.d. <2%. (B) No differences in the cycling of both ERα isoforms on the pS2 promoter are evidenced in kinetic ChIP assays performed using chromatin prepared from MDA-MB231 cells expressing either ERα isoforms. Next, experiments probed the functional impact of these cycles of ERα isoforms binding in MDA∷ERα66 (C) and MDA∷ERα46 cells (D). These assays evaluated the recruitment and activation of P-PolII in response to 10−8 M estradiol (E2) added at times indicated within the graphs (A–C of each panels).
Figure 4
Figure 4
Identification of the transcription factors targeted by apo-ERα isoforms on the pS2 promoter in vivo. (A) Real-time PCR-mediated quantification of ChIP experiments performed using chromatin prepared from MCF-7 cells either treated with ethanol as vehicle control (EtOH) or firstly synchronized by α-amanitin treatment. Comparing the results obtained in both conditions enables determination of proteins directly recruited by both apo-ERα isoforms on the pS2 promoter (those after α-amanitin treatment, in gray). (B) Comparative ChIP assays using chromatin prepared from MCF-7, MDA-MB231 or MDA-MB231 cells expressing either ERα isoforms.
Figure 5
Figure 5
Re-ChIP screening of the factors recruited to the pS2 promoter by apo-ERα isoforms. Chromatin prepared from α-amanitin synchronized MDA-MB231 cells expressing ERα66 (A) or ERα46 (B) was subject to the ChIP procedure with the antibodies shown at the left side, and again immunoprecipitated using antibodies shown at the top of the panels. (C) Scheme illustrating the topology of the complexes recruited to the pS2 promoter, as indicated through the comprehensive Re-ChIP analysis shown above.
Figure 6
Figure 6
Kinetics of the recruitment of transcription factors directed by apo-ERα isoforms on the pS2 promoter. (A, B) Kinetic ChIP experiments were performed and expressed as in Figure 4, using antibodies and chromatin prepared from synchronized MDA-MB231 cells expressing either ERα isoforms, as indicated within the graphs. (C) Scheme summarizing the sequence of recruitment of transcription factors directed by ERα66 and ERα46 onto the pS2 promoter. The numbering reflects the order of engagement of the different complexes onto the promoter. In the case of ERα46, V-a and V-b are combinatorial steps.
Figure 7
Figure 7
Dynamic chromatin modifications within the pS2 promoter. (A) Kinetic ChIP experiments were performed as depicted in Figure 4, using antibodies and chromatin prepared from synchronized MDA-MB231 cells expressing either ERα isoforms, as indicated within the graphs. (B) Schematic representation of the pS2 promoter, with phased nucleosomes and regions amplified by PCR primers depicted. (C) Mononucleosomes prepared from indicated cells were used in ChIP assays detecting modified histones in both NucE and NucT. The absence of amplification using the B and D primer pairs controls proper digestion. (D) Fluctuations in NucE and NucT positions on the pS2 promoter were assessed through PCR amplification of mononucleosomes using the A to J primer pairs. (E) Following α-amanitin synchronization, mononucleosomes were prepared at times indicated and subjected to ChIP and Re-ChIP procedures using the indicated antibodies. Use of the E and H primer pairs for DNA amplification showed the localization of the modified histones within either NucE or NucT. (F) Scheme summarizing the dynamic histone modifications and nucleosomal organization of the pS2 promoter during ERα isoforms cycles. Dashed lines in NucT reflect the increased accessibility of the TATA box surrounding region for MNase digestion. The recruitment events that are hypothesized to correlate with the observed nucleosome modifications are boxed and highlighted.
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References

    1. Barkhem T, Haldosén LA, Gustafsson JA, Nilsson S (2002) Transcriptional synergism on the pS2 gene promoter between a p160 coactivator and ERα depends on the coactivator subtype, the type of ERE, and the promoter context. Mol Endocrinol 16: 2571–2581 - PubMed
    1. Bauer UM, Daujat S, Nielsen SJ, Nightingale K, Kouzarides T (2002) Methylation at arginine 17 of histone H3 is linked to gene activation. EMBO Rep 31: 39–44 - PMC - PubMed
    1. Belandia B, Parker MG (2003) Nuclear receptors: a rendezvous for chromatin remodeling factors. Cell 114: 277–280 - PubMed
    1. Bian Z, Nilsson S, Gustafsson JA (2001) Selective estrogen receptor modulators and coronary heart disease. Trends Cardiovasc Med 11: 196–202 - PubMed
    1. Bird A (2002) DNA methylation patterns and epigenetic memory. Genes Dev 16: 6–21 - PubMed

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