1. Volume 31, Issue 2, Pages 212-221 (July 2008)
Regulation of Estrogen Rapid Signaling through Arginine Methylation by PRMT1 
Muriel Le Romancer, Isabelle Treilleux, Nicolas Leconte, Yannis Robin-Lespinasse, Stéphanie Sentis, Katia Bouchekioua-Bouzaghou, Sophie Goddard, Stéphanie Gobert-Gosse, Laura Corbo 
Molecular Cell 
Volume 31, Issue 2, Pages (July 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1 PRMT1 Specifically Methylates ERα In Vitro
(A) ERα domains and putative PRMT1 methylation sites: “A/B,” encompassing Activation Function-1 (AF-1); “C,” containing the DNA binding domain (DBD); “D,” containing nuclear localization signals (NLS); “E,” containing the ligand-bind domain (LBD) and Activation Function-2 (AF-2); and finally, the agonists/antagonists regulatory “F” region.
(B) Purified GST-ERα fragments encompassing the RGG motifs -ER and -ER were incubated with recombinant GST-PRMT1 or His-CARM1, in the presence of [methyl-3H] SAM. Recombinant histone H4 (H4) and histone H3 (H3) were used as positive controls and GST as a negative control. Reaction products were analyzed by SDS-PAGE followed by fluorography.
(C) Purified GST-ERα fragments containing -ER and -ER were assayed for methylation by GST-PRMT1 as described in (B).
(D) Methylation assay with -ER (wild-type) and -ER (R260A) (mutant) ERα fragments as described in (B).
(E) Methylation assay with GST constructs -ER (wild-type) and -ER (R260A) (mutant) using GST-PRMT1 in the presence or in the absence of 0.5 mM SAM. Reaction products were analyzed by SDS-PAGE followed by western blotting with the anti-ERα(CH3)R260 antibody (upper panel). Coomassie blue staining of the gel (lower panel).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 PRMT1 Methylates ERα on R260 In Vivo
(A) E2 induces ERα methylation in MCF-7 cells. Immunoprecipitation of methylated endogenous ERα from extracts of estrogen-deprived MCF-7 cells (t = 0) stimulated with 10−8 M estradiol for the indicated times was performed with the anti-ERα(CH3)R260 antibody, followed by western blotting with antibody against ERα.
(B) ERα methylation is inhibited by the general inhibitor of methylation, AdOx. Estrogen-deprived MCF-7 cells were incubated or not with 40 μM AdOx for 24 hr, treated with vehicle or E2 (10−8 M) for 5 min, lysed and immunoprecipitated with the anti-ERα(CH3)R260 antibody, and revealed as in (A).
(C) PRMT1 is responsible for ERα methylation in response to E2 induction. Lysates of MCF-7 cells transfected with control siRNA duplexes or with specific PRMT1 siRNA duplexes were tested for ERα methylation as in (A).
(D) The anti-ERα(CH3)R260 antibody specifically recognizes ERα methylated on R260. HeLa cells transfected with wild-type or mutated (R260A) ERα, and PRMT1-expressing plasmids were grown on charcoal-stripped serum and treated with estrogens for the indicated times. The methylation status of wild-type and mutated ERα was analyzed as above.
ERα, PRMT1, and GAPDH in inputs are shown.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Methylation of ERα Occurs in the Cytoplasm of MCF-7 Cells
(A) Estrogen-deprived MCF-7 cells were treated with E2 (10−8 M) for 5 min, and cytoplasmic (Cyt) and nuclear (NE) fractions were prepared. The methylation level of ERα was analyzed by immunoprecipitation with the anti-ERα(CH3)R260 followed by western blot with anti-ERα antibody.
(B) Estrogen-deprived MCF-7 cells (t = 0) were stimulated with 10−8 M estradiol for the indicated times. Cytoplasmic and nuclear fractions were prepared at each time, then immunoprecipitated with anti-ERα(CH3)R260 followed by western blotting with anti-ERα antibody. The nuclear extracts were also precipitated with anti-ERα antibody (used as IP control).
(C) MCF-7 cells were treated or not with the proteasome inhibitor MG132 (10 μM) 6 hr before E2 treatment, then incubated with the vehicle or E2 for the indicated times. Cytoplasmic and nuclear fractions prepared as in (B) were immunoprecipitated with anti-ERα(CH3)R260 followed by western blotting with anti-ERα antibody as in (B).
The quality of the fractionation was evaluated by western blot with the anti-ERα and anti-GAPDH antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Methylated ERα Is Essential for E2-Induced Assembly of ERα/Src/p85: Crosstalk between Methylated ERα and Src Activity
(A) p85 and Src coimmunoprecipitate with methylated ERα. Lysates of MCF-7 cells transfected with control siRNAs or with specific PRMT1 siRNAs were tested for ERα methylation. The immunoprecipitates were blotted with anti-p85 and anti-Src antibodies. The amount of ERα, p85, Src, and PRMT1 in the different samples was determined by western blot.
(B) PRMT1 is essential to the assembly of the ERα/Src/PI3K complex. Lysates of MCF-7 cells transfected with control siRNAs or with specific PRMT1 siRNAs as in (A) were treated with vehicle or E2 (10−8 M), immunoprecipitated with the anti-Src antibody, and analyzed with the indicated antibodies.
(C) p85 interacts only with methylated ERα. MCF-7 cells treated with E2 as above were lysed and immunoprecipitated with anti-ERα(CH3)R260 antibody, followed by western blotting with anti-p85 and anti-ERα antibodies. Unbound fractions were then used for a second immunoprecipitation using anti-ERα antibody followed by western blotting with anti-p85. The amount of ERα immunoprecipitated is also shown.
(D) Src activity is required for E2-induced ERα methylation. MCF-7 cells were treated or not with PP1 (5 μM) 15 min before E2 treatment, then incubated with the vehicle or E2 for the indicated times. ERα methylation status was evaluated as described above. ERα expression in inputs is shown (lower panel).
(E) Focal adhesion kinase (FAK) is present in the E2-induced complex with ERα/Src/p85. Cell lysates from MCF-7 cells treated as in (D) were immunoprecipitated and analyzed using the indicated antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
E2 Activation of Akt Is Mediated by Methylated ERα: Role of PRMT1 on Cell-Cycle Progression
(A) NIH 3T3 fibroblasts were transfected with wild-type or mutated (R260A) ERα-expressing plasmids. Estrogen-deprived cells (t = 0) were stimulated with 10−8 M estradiol for the indicated times. Lysates were analyzed for P-Akt (Ser473) (upper section). Akt and ERα expression in inputs was evaluated by western blotting.
(B) Tamoxifen inhibits E2-induced Akt phosphorylation in MCF-7 cells. MCF-7 cells were stimulated with E2 (10−8 M) or with E2 plus tamoxifen (10−6 M) for the indicated times. Akt phosphorylation was analyzed as in (A).
(C) PRMT1 is involved in E2-dependent Akt activation. MCF-7 lysates of cells transfected with control or with specific PRMT1 siRNA duplexes as described in Figure 3B were analyzed for P-Akt (Ser473) (upper section). The same membrane was reprobed with anti-Akt antibody.
(D) PRMT1 regulates cell proliferation. MCF-7 cells transfected with control siRNA or PRMT1 siRNAs were analyzed for expression of Cyclin D1 and p21. PRMT1 and GAPDH levels were measured as controls (left panel). Cell-cycle distribution was determined by FACS analysis of propidium iodine-stained cells.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Expression of ERα in Breast Cancer Tissues
(A) Methylated ERα localizes in the cytoplasm of tumor cells (high level of expression) and in the normal counterpart (low level of expression), but not in myoepithelial cells [anti-ERα(CH3)R260; objective, ×40].
(B) Low (Ba) and high expression levels (Bb and Bc) of methylated ERα in infiltrating breast cancers. Similar expression levels in ERα-positive (Bb) and ERα-negative (Bc) tumors using anti-ERα(CH3)R260 (objective, ×40).
(C) Expression of methylated and nonmethylated ERα in two serial sections from the same tumor using anti-ERα(CH3)R260 (cytoplasmic staining) (left panel) and anti-ERα 1D5 (right panel) antibodies that recognize both methylated ERα (cytoplasmic staining) and nonmethylated ERα (nuclear staining). (Ca and Cb) ERα-negative tumor expressing only methylated ERα. (Cc and Cd) ERα-positive tumor expressing both nonmethylated and methylated ERα. (Ce and Cf) ERα-positive tumor expressing only nonmethylated ERα. Objective, ×40.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Model of the Assembly and Regulation of the ERα Macromolecular Complex Involved in Nongenomic Signaling
In the absence of hormone, ERα, p85, Src, and FAK are not associated (1). E2 induces ERα methylation via PRMT1 and partial FAK dephosphorylation, triggering Src/FAK interaction (2). These two events are necessary for the association of ERα, Src, p85, and FAK in a macromolecular complex (3). The complex formation elicits Akt activation that regulates cellular processes. Then, ERα is demethylated by a putative arginine demethylase that disrupts the main complex (4).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions