Estrogen receptor β (ERβ1) transactivation is differentially modulated by the transcriptional coregulator Tip60 in a cis-acting element-dependent manner

Abstract

Estrogen receptor (ER) β1 and ERα have overlapping and distinct functions despite their common use of estradiol as the physiological ligand. These attributes are explained in part by their differential utilization of coregulators and ligands. Although Tip60 has been shown to interact with both receptors, its regulatory role in ERβ1 transactivation has not been defined. In this study, we found that Tip60 enhances transactivation of ERβ1 at the AP-1 site but suppresses its transcriptional activity at the estrogen-response element (ERE) site in an estradiol-independent manner. However, different estrogenic compounds can modify the Tip60 action. The corepressor activity of Tip60 at the ERE site is abolished by diarylpropionitrile, genistein, equol, and bisphenol A, whereas its coactivation at the AP-1 site is augmented by fulvestrant (ICI 182,780). GRIP1 is an important tethering mediator for ERs at the AP-1 site. We found that coexpression of GRIP1 synergizes the action of Tip60. Although Tip60 is a known acetyltransferase, it is unable to acetylate ERβ1, and its coregulatory functions are independent of its acetylation activity. In addition, we showed the co-occupancy of ERβ1 and Tip60 at ERE and AP-1 sites of ERβ1 target genes. Tip60 differentially regulates the endogenous expression of the target genes by modulating the binding of ERβ1 to the cis-regulatory regions. Thus, we have identified Tip60 as the first dual-function coregulator of ERβ1.

Keywords: AP-1; Acetyltransferase; Coregulator; ERβ1; Estrogen Receptor; Estrogen-response Element; Protein-Protein Interactions; Transcription; Transcription Enhancers; Transcription Repressor.

FIGURE 1.

ERβ1 can interact with Tip60 in either the absence or presence of estrogen. A, Tip60 interacts with ERβ1 and ERα in vitro. ERβ1, ERα, and HA-tagged Tip60 were translated in vitro and labeled with [³⁵S]methionine. The lysates were mixed and incubated with E₂ and then immunoprecipitated (IP) with HA antibody. The immunoprecipitates were resolved by SDS-PAGE and analyzed by autoradiography. B, ERβ1 interacts with Tip60 in yeast cells independent of E₂. ERβ1, ERα, or empty vector (pGBKT7) was transformed into yeast with Tip60. The transformed cells were grown on quadruple dropout agar (QDO) containing X-α-galactosidase and DMSO or E₂ until the appearance of blue colonies. C, ERβ1 interacts with Tip60 in vivo. HEK293 cells were grown in CSS-containing medium and transfected with ERβ1 and His-tagged Tip60 before the addition of E₂. Lysates were precipitated on an Ni-NTA column and immunoblotted (IB) with ERβ1 or Tip60 antibody. The samples were run on the same gel. D, ERβ1-Tip60 interaction was confirmed by reciprocal coimmunoprecipitation. Procedures were similar to those in C, except that lysates were immunoprecipitated with ERβ1 antibody. E, ERβ1 interacts with Tip60 in an E₂-independent manner in a hormone-sensitive prostate cancer cell line, PC-3. ERβ1 stably expressed PC-3 cells (PC-3-ERβ1) were grown in CSS-containing medium before the addition of DMSO or E₂. Lysates were immunoprecipitated by ERβ1 antibody and immunoblotted with ERβ1 or Tip60 antibody. F, ERβ1 colocalized with Tip60 with or without E₂. HEK293 cells were grown in CSS-containing medium transfected with ERβ1 and Tip60 followed by the incubation of DMSO (vehicle) (upper panel) or E₂ (lower panel). G, ERβ1 colocalized with Tip60 in PC-3. PC-3-ERβ1 cells were grown in full-serum containing medium. F and G, antibodies to ERβ1 and Tip60 were used for immunostaining, and DAPI was used as the nuclear marker. The images in F and G were captured by a fluorescence microscope. Bar, 20 μm.

FIGURE 2.

Hinge domain of ERβ1 is responsible for the interaction with Tip60. A, schematic diagram shows the domains of full-length ERβ1 and different domain-deleted constructs. The c-Myc tag was added to the N terminus of each construct. The strength of interaction between different ERβ1 constructs and Tip60 is represented by “+” and “−” signs. “+++” represents the strongest interaction, and “−” represents no interaction. AF-1, activation function 1; AF-2, activation function 2. B, HEK293 cells were grown in CSS-containing medium and transfected with Tip60 and different domain-deleted ERβ1 constructs. Lysates were immunoprecipitated (IP) with c-Myc antibody. Immunoglobulin IgG was used as the negative control. The immunoprecipitates were immunoblotted (IB) with c-Myc or Tip60 antibody. Asterisks denote the positions of ERβ1 and its mutants.

FIGURE 3.

Tip60 differentially regulates ERβ1 transactivation at ERE and AP-1 sites but has minimal effect on other transcription factor-binding sites. A–E, Tip60 reduces ERβ1 transactivation at various ERE sites. ERβ1 was transfected with GFP or Tip60 together with pCMV-β-gal and vitellogenin ERE (A), C3 ERE (B), c-Fos ERE (C), pS2 ERE (D), or progesterone receptor ERE (E) reporter plasmids into HEK293 cells grown in CSS-containing medium. F, inhibition of ERβ1 transactivation by Tip60 is concentration-dependent. ERβ1 was transfected with different amounts of GFP and Tip60 together with pCMV-β-gal and vitellogenin ERE reporter plasmid. Different ratios of plasmids of Tip60 to GFP were transfected. G–I, Tip60 enhances ERβ1 transactivation at AP-1 sites but has minimal effect on other transcription factor-binding sites. ERβ1 was transfected with GFP or Tip60 together with pCMV-β-gal and reporter plasmids containing the binding site of AP-1 (G), NFκB (H), or Sp1 (I) into HEK293 cells grown in CSS-containing medium. J–M, Tip60 reduces ERβ1 transactivation at ERE site but increases its transactivation at AP-1 site in different PCa cell lines. ERβ1 was transfected with GFP or Tip60 together with pCMV-β-gal and reporter plasmids containing vitellogenin ERE (J and L) or AP-1-binding site (K and M) into PC-3 or DU-145 cells grown in CSS-containing medium. After the transfection, HEK293, PC-3, and DU-145 cells were added with DMSO or E₂. Relative luciferase activity was determined and normalized with the β-gal activity. Results were the average of three independent experiments. All data are represented as mean ± S.D. The statistical significance of the difference in luciferase activity between the overexpression of GFP and Tip60 in the presence of DMSO or E₂ is shown as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

Various ligands modulate Tip60-mediated regulatory effects on ERβ1 transactivation. A and B, ERβ1 was transfected with GFP or Tip60 together with pCMV-β-gal and vitellogenin ERE (A) or AP-1 reporter plasmids (B) into HEK293 cells grown in CSS-containing medium. Various ligands, namely E₂ (10 nm), DPN (10 nm), GEN (1 μm), EQ (1 μm), DAI (1 μm), API (100 nm), TAM (1 μm), RAL (1 μm), ICI (1 μm), and BPA (10 nm), were added, respectively, or DMSO was used as the control after transfection for 24 h. Relative luciferase activity was determined as above. The results are the average of at least two independent experiments. All data are represented as mean ± S.D. The statistical significance of the difference in luciferase activity between the overexpression of GFP and Tip60 in the presence of each ligand is shown as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

ERβ1 cannot be acetylated by Tip60 and preferentially interacts with unacetylated Tip60. A, schematic diagram shows the structural domains of Tip60 and the substitution of amino acids on the HAT-defective mutant (Q377E/G380E) (Tip60ΔHAT). B, ERβ1 is not acetylated by Tip60 in vitro. His-tagged wild-type of Tip60 (Tip60WT) or Tip60ΔHAT was transfected, respectively, into HEK293 cells, and Tip60 proteins were purified on an Ni-NTA column. Recombinant ERβ1 protein and Tip60 were incubated in HAT buffer containing acetyl-CoA. The immunoprecipitates (IP) were immunoblotted (IB) with acetyl-lysine, ERβ1, or Tip60 antibody. Asterisk denotes the nonspecific band that appeared when the blot was immunoblotted with pan-acetyl-lysine antibody. C, ERβ1 is not acetylated by Tip60 in vivo and preferentially interacts with unacetylated Tip60. Tip60WT or HAT was transfected with ERβ1 into HEK293 cells. Lysates were immunoprecipitated with either ERβ1 (left panel) or Tip60 (middle panel) antibody. Immunoglobulin IgG was used as the negative control. The immunoprecipitates were immunoblotted with acetyl-lysine, ERβ1, or Tip60 antibody.

FIGURE 6.

HAT activity of Tip60 is not necessary for regulation of the ERβ1 transactivation at AP-1 and ERE sites. A, expression of Tip60ΔHAT was similar to that of Tip60WT. Lysates were extracted and immunoblotted (IB) with Tip60 antibody. β-Actin was used as the loading control. B and C, HAT activity of Tip60 is not necessary for the regulation of ERβ1 transactivation at AP-1 and ERE sites. GFP, Tip60WT, or Tip60ΔHAT was transfected, respectively, with ERβ1, pCMV-β-gal (B), AP-1(C), or vitellogenin-ERE reporter plasmids into HEK293 cells before the addition of E₂. D and E, GFP or Tip60 was transfected, respectively, with ERβ1, pCMV-β-gal (D), AP-1 (E), or vitellogenin-ERE reporters into HEK293 cells. After the transfection, DMSO or E₂ together with ethanol (vehicle) or anacardic acid (AnAc) was added as indicated. B–D, relative luciferase activity was determined as in Fig. 3. Results are the average of three independent experiments. Data are represented as mean ± S.D. The statistical significance of the difference in luciferase activity between the overexpression of GFP and Tip60 in the presence of DMSO or E₂ is shown as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 7.

Tip60 interacts with GRIP1 to enhance ERβ1 transactivation at the AP-1 site synergistically. A and B, Tip60 and GRIP1 exert a synergistic effect on ERβ1 transactivation at the AP-1 site. Different combinations of GFP, Tip60, GRIP1, and SRC1 were transfected with ERβ1, pCMV-β-gal, vitellogenin-ERE (A) or AP-1 reporter plasmids (B) as indicated. After the transfection, DMSO or 10 nm E₂ was added as indicated. C, synergistic effect of Tip60 and GRIP1 on the ERβ1 transactivation at the AP-1 site is concentration-dependent. GFP or different ratios of plasmids of Tip60 to GRIP1 were transfected. DMSO was added after the transfection. Relative luciferase activity was determined as in Fig. 3. Results are the average of three independent experiments. Data are presented as mean ± S.D. The statistical significance of the difference in luciferase activity between overexpressing Tip60, GRIP1, and GFP is shown as *, p < 0.05; **, p < 0.01; ***, p < 0.001. D, Tip60 forms a multiprotein complex with p160 coactivators and ERβ1. HEK293 cells were transfected with Tip60, ERβ1, SRC1, and GRIP1 and grown in CSS-containing medium. Lysates were immunoprecipitated (IP) with Tip60 antibody. Immunoglobulin IgG was used as the negative control. The immunoprecipitates were immunoblotted (IB) with Tip60, ERβ1, GRIP1, or SRC1 antibody as indicated.

FIGURE 8.

Tip60 differentially regulates ERβ1 target genes possessing ERE or AP-1 sites at their cis-regulatory regions in PC-3 cells. A and B, expression of ERβ1 and Tip60 in ERβ1 and LacZ stably expressed PC-3 cells upon the knockdown of Tip60 was determined. PC-3-LacZ/-ERβ1 cells were grown in CSS-containing medium and transfected with nontargeting control siRNA (siNT) or siRNAs specific to Tip60 (siTip). E₂ was added after 24 h. Expression of ERβ1 (A) and Tip60 (B) was determined by quantitative RT-PCR. Human GAPDH was used as the housekeeping gene. C and D, Tip60 differentially regulates ERβ1 target genes. PC-3-LacZ/-ERβ1 cells were treated as described in A and B. Expression of CXCL12 (C) and cyclin D2 (D) was determined by quantitative RT-PCR. The results are the average of three independent experiments. All data are represented as mean ± S.D. The statistical significance of the difference in gene expression between different treatments is shown as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001. E–G, ERβ1 and Tip60 are both recruited to the cis-regulatory regions of CXCL12 and cyclin D2. PC-3-ERβ1 cells were grown in CSS-containing medium added with E₂. ChIP assays were performed with ERβ1 (E) or Tip60 antibody (F). G, re-ChIP assay was performed with Tip60 antibody followed by the second immunoprecipitation with ERβ1 antibody. The ChIP DNA was amplified by real time PCR for the target regions containing an ERE site of CXCL12 or an AP-1 site of cyclin D2. The genomic region of ERβ isoform 5 (ERβ5) containing neither an ERE nor an AP-1 site was used as the negative control. The fold enrichment of recruitment of ERβ1 and/or Tip60 at the target regions is relative to respective IgG controls. The results are the average of two independent experiments. All data are represented as mean ± S.D. The statistical significance of the difference in the recruitment between ERβ1 (and/or Tip60) and IgG is shown as follows: *, p < 0.05. H, Tip60 differentially regulates the recruitment of ERβ1 to the cis-regulatory regions of CXCL12 and cyclin D2. PC-3-ERβ1 cells were grown in CSS-containing medium added with E₂ and transfected with siRNAs (siNT or siTip) for 48 h. ChIP assays were performed with ERβ1 antibody. The procedures of the amplification of ChIP DNA were similar to those described in E–G. The results are the average of two independent experiments. Data are represented as mean ± S.D. The statistical significance of the difference in the ERβ1 recruitment with or without the knockdown of Tip60 is shown as follows: *, p < 0.05.