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. 2011 Jun;152(6):2517-26.
doi: 10.1210/en.2010-1281. Epub 2011 Apr 19.

Phosphorylation of Estrogen Receptor α at serine 118 directs recruitment of promoter complexes and gene-specific transcription

Tamika T Duplessis  1 , Christopher C Williams, Steven M Hill, Brian G Rowan
Affiliations

Phosphorylation of Estrogen Receptor α at serine 118 directs recruitment of promoter complexes and gene-specific transcription

Tamika T Duplessis et al. Endocrinology. 2011 Jun.

Abstract

Phosphorylation of estrogen receptor α (ERα) is important for receptor function, although the role of specific ERα phosphorylation sites in ERα-mediated transcription remains to be fully evaluated. Transcriptional activation by ERα involves dynamic, coordinate interactions with coregulators at promoter enhancer elements to effect gene expression. To determine whether ERα phosphorylation affects recruitment of unique protein complexes at gene-specific promoters, changes in ERα Ser118 phosphorylation were assessed for effects on receptor and coregulator recruitment and transcription of ERα-regulated genes. Chromatin immunoprecipitation assays to measure promoter association found a 17β-estradiol (E2)-dependent recruitment of ERα at 150 min to ERα-regulated promoters, whereas ERα phosphorylated at Ser118 was dissociated from promoters after E2 treatment. Mutation of Ser118 to alanine (S118A) altered unliganded and ligand-induced association of ERα and p160 coregulators with ERα target promoters when compared with wild-type (WT)-ERα transfection. S118A and WT-ERα exhibited a similar level of recruitment to the estrogen response element-driven pS2 promoter and induced pS2 mRNA after E2 treatment. Although WT-ERα was recruited to c-myc and cyclin D1 promoters after E2 treatment and induced mRNA expression, S118A exhibited reduced interaction with c-myc and cyclin D1 promoters, and E2 did not induce c-myc and cyclin D1 mRNA. In addition, S118A resulted in increased recruitment of steroid receptor coactivator-1, glucocorticoid receptor interacting protein-1, and activated in breast cancer-1 to pS2, c-myc, and cyclin D1 irrespective of the presence of E2. Together, these data indicate that site specific phosphorylation of ERα directs gene-specific recruitment of ERα and transcriptional coregulators to ERα target gene promoters.

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Figures

Fig. 1.
Fig. 1.
Time-dependent, 17B-estradiol (E2)-induced changes of ERα Ser118 phosphorylation status in MCF-7 cells. A, MCF-7 cell extracts were immunoblotted with antibodies against pan-ERα (ERα) and phospho-ERα S118 after incubation with 10−8 m E2 as described in Materials and Methods. GAPDH served as a loading control. Levels of (B) P-ERα Ser118 and (C) ERα were quantified by densitometry. These data are representative of three or more independent experiments that yielded comparable results. a, P < 0.05 compared with time 0 min.
Fig. 2.
Fig. 2.
ERα P-S118 recruitment to different classes of estrogen responsive promoters. MCF-7 cells were plated in 10% charcoal-stripped serum and grown to 70% confluency before serum depravation overnight. Cells were then incubated with vehicle (mock stimulated) or 10−8 m E2 for 150 min at 37 C after α-amanatin release. Chromatin was prepared and immunoprecipitated with antibodies against pan-ERα (ERα), P-ERα Ser118, and corresponding species-specific IgG control. Relative levels of (A) pS2 ERE, (B) c-myc AP-1, or (C) cyclin D1 CRE promoter interaction were measured by real-time PCR and were normalized to input. Data reported details one representative experiment from at least five independent experiments ± sd. a, P < 0.05 compared with IgG; b, P < 0.05 compared with vehicle treatment. Ctrl, Control.
Fig. 3.
Fig. 3.
Effects of site-directed mutation of S118 on ERα-dependent gene expression. A, HeLa cells were cotransfected with ERE2-TK-Luc reporter and either wild-type ERα or ERα S118 mutant expression plasmids (ERα S118A or ERα S118E) as indicated in the figure. B, At 24 h after transfection, cells were incubated with vehicle (mock stimulated) or 10−8 m E2 24 h. Standard luciferase assays were performed on cell extracts in triplicate as described in Materials and Methods. Each bar represents the averages of at least three independent experiments ± sd. a, P < 0.05 compared with WT-ERα-transfected groups incubated with vehicle; b, P < 0.05 compared with WT-ERα-transfected groups incubated with E2. C, HeLa cells were transfected with WT-ERα ERα, ERα S118A, and ERα S118E; 24 h after transfection, cells were incubated with vehicle (mock stimulated) or 10−8 m E2 for 3 h at 37 C. mRNA expression of pS2, c-myc, and cyclin D1 was measured by real-time RT-PCR as described in Materials and Methods. ERα protein was measured by Western blotting normalized α-tubulin. mRNA expression was normalized to β-actin, and each bar represents the mean of three independent experiments ± sd. a, P < 0.05 compared with vehicle.
Fig. 4.
Fig. 4.
Effects of site directed mutation of S118 on ERα and p160 coactivator association with ERα-target promoters. HeLa cells were transfected with either wild-type ERα or ERα S118A expression plasmids; 24 h after transfection, cells were incubated with vehicle (mock stimulated) or 10−8 m E2 for 2.5 h at 37 C after α-amanatin release. Chromatin was prepared and immunoprecipitated with antibody against pan-ERα (ERα), SRC-1, GRIP-1, AIB-1, and corresponding species-specific IgG control antibodies. Relative levels of (A) pS2 ERE, (B) c-myc AP-1, or (C) cyclin D1 CRE promoter interaction were measured by real-time RT-PCR and were normalized to input. Data are representative of at least four independent experiments ± sd. a, P < 0.05 compared with IgG; b, P < 0.05 compared with vehicle. D, Table summary of A–C.
Fig. 5.
Fig. 5.
Summary of ERα and coactivator recruitment and gene expression in cells transfected with WT-ERα or ERα S118A. A, Promoter occupancy of pS2 ERE. In WT-ERα transfected cells, SRC-1 and GRIP-1 are recruited; however, E2 disrupted the association of these proteins. SRC-1, GRIP-1, and AIB-1 are recruited with ERα S118A. These interactions remained intact with the addition of E2. B, Promoter occupancy of c-myc AP-1. In the absence of ligand, SRC-1 was recruited to c-myc AP-1, and the addition of E2 disrupted this association in both WT-ERα and ERα S118A-transfected cells. C, Promoter occupancy of cyclin D1 CRE. In the absence of ligand, no specific recruitment of p160 coregulators was observed with WT-ERα. Cells transfected with ERα S118A resulted in recruitment of SRC-1 and GRIP-1. In cells transfected with ERα S118A, the addition of E2 resulted in decreased associations of ERα and SRC-1 but increased recruitment of GRIP-1 and AIB-1. Solid borders around proteins indicate protein recruitment to the promoter; dashed borders around proteins represents promoter dissociation. AFT-2, Activating transcription factor-2; Jun, Jun proto-oncogene; Fos, FBJ muring osteosarcoma viral oncogene.
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