Aryl hydrocarbon receptor-mediated transcription: ligand-dependent recruitment of estrogen receptor alpha to 2,3,7,8-tetrachlorodibenzo-p-dioxin-responsive promoters

Abstract

Using chromatin immunoprecipitation assays, we studied the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated recruitment of the aryl hydrocarbon receptor (AhR) and several co-regulators to the CYP1A1 promoter. AhR displayed a time-dependent recruitment, reaching a peak at 75 min and maintaining promoter occupancy for the remainder of the time course. Recruitment of AhR was followed by TIF2/SRC2, which preceded CBP, histone H3 acetylation, and RNA polymerase II (RNAPII). Simultaneous recruitment to the enhancer and the TATA box region suggests the formation of a large multiprotein complex bridging the two promoter regions. Interestingly, estrogen receptor alpha (ERalpha) displayed a TCDD- and time-dependent recruitment to the CYP1A1 promoter, which was increased by co-treatment with estradiol. Transfection in HuH7 human liver cells confirmed previously reported ERalpha enhancement of AhR activity. In contrast, TCDD did not induce the recruitment of ERalpha to the estrogen-responsive pS2 promoter, and after 120 min of co-treatment with estradiol, ERalpha is still present on the CYP1A1 promoter but no longer at pS2. RNA interference studies with T47D cells support a role for ERalpha in TCDD-dependent CYP1A1 expression. Our data suggest that ERalpha acts as a coregulator of AhR-mediated transcriptional activation and that the recruitment of ERalpha by AhR represents a novel mechanism AhR-ERalpha cross talk.

FIG.1.

Temporal recruitment of AhR and associated factors to CYP1A1 regulatory regions. (A) MCF-7 human breast carcinoma cells were treated with 10 nM TCDD for the specified amounts of time (minutes). ChIP assays were performed as described in Materials and Methods with antibodies against the indicated proteins. PCRs of DNA from the input or immunoprecipitated fractions were performed using primer pairs that amplify the CYP1A1 promoter regions as indicated. (B) ChIP assay samples were analyzed by real-time PCR, and the results were normalized to time zero (no ligand). RNApol, RNA polymerase. (C) Genomic DNA and the sonicated input DNA were PCR amplified using the specified primer pairs. The sonicated input fraction was separated by 0.7% agarose gel electrophoresis and visualized by ethidium bromide staining. (D) ChIP assays with antibodies to the indicated proteins were analyzed by PCR using primer pairs covering the specified regions of CYP1A1. IgG, immunoglobulin G.

FIG. 2.

Ligand-dependent recruitment of ERα to AhR target genes. (A) MCF-7 cells were treated with 10 nM estradiol (E2), 10 nM TCDD, 10 nM TCDD plus 10 nM E2, or DMSO (0.1%) for the indicated amounts of time. ChIP assays were performed with primer pairs specific to the XRE enhancer regions of CYP1A1 using conventional PCR (A) and real-time PCR (B). ChIP analysis of the promoter occupancy of the CYP1B1 promoter by AhR and ERα was done using conventional PCR (C) and real-time PCR (D). T47D cells were treated with 10 nM TCDD or 10 nM TCDD plus 10 nM E2, followed by ChIP and real-time PCR amplification of the CYP1A1 (E) and CYP1B1 (F) enhancer regions. HuH7 human hepatoma cells were transfected with pSG5-ERα or a vector control and treated as described for panel A for the specified amounts of time. ChIP assays were performed with primer pairs specific to the XRE-containing enhancer region of CYP1A1 using conventional PCR (G) and real-time PCR (H). Results shown are representative of at least two independent experiments. ERα antibodies used were H-184 (ERα*) and HC-20 (ERα**). IgG, immunoglobulin G.

FIG. 3.

Effect of ERα on AhR-mediated transcription. HuH7 human hepatoma cells were transiently transfected as described in Materials and Methods. Following transfection, cells were treated with the indicated compounds for 24 h prior to assaying for luciferase and β-galactosidase activities. Cells were transiently cotransfected with increasing amounts of pSG5-hERα and either p1A1LUC-containing the CYP1A1 promoter (A) or p3xXRE-LUC (B). Results are expressed as the means and standard deviations of four replicate determinations. Reporter gene activity significantly (P < 0.05) greater than the TCDD-treated vector control is indicated by an asterisk, reporter gene activity significantly (P < 0.05) greater than TCDD-treated samples transfected with the same amounts of ERα is indicated by a dagger, and reporter gene activity significantly (P < 0.01) greater than TCDD-1 nM E2-treated samples transfected with the same amounts of ERα is indicated by a double dagger. (C) Cells were cotransfected with p1A1-LUC and one of the following constructs: pSG5 (vector), pSG5-hERα (ERα), pSG5-hERβ (ERβ), pSG5-hERα-ΔAF1 (ERα ΔAF1), pSG5-HEO (ERα HEO), or pSG5-HEO S118A (HEO S118A). Reporter gene activity significantly (P < 0.05) different than the TCDD- or TCDD+E2-treated vector control is indicated by an asterisk, and reporter gene activity significantly (P < 0.05) greater than TCDD-treated samples transfected with the same ER plasmids is indicated by a number sign. (D) Cells were transiently cotransfected with p1A1LUC and either pSG5 or pSG5-hERα (ERα). Cells were treated for 24 h with a vehicle control or 10 nM TCDD or cotreated with 10 nM TCDD and 10 nM E2, 100 nM ICI 182,780, 100 nM 4-hydoxytamoxifen (TAM), or 100 nM raloxifene (RAL). Reporter gene activity significantly (P < 0.05) greater than the TCDD-treated vector control is indicated by an asterisk, and reporter gene activity significantly (P < 0.05) different than the TCDD-treated sample transfected with the ERα is indicated by a number sign. Results are representative of at least two independent experiments and are expressed as the means and standard deviations of four replicate determinations.

FIG. 4.

Real-time PCR results of the effect of ERα and estradiol on transcription at the CYP1A1 promoter. Total RNA isolated from MCF-7 cells (A, B), T47D cells (C, D), and HuH7 cells (E, F) transfected with or without ERα, treated with 10 nM TCDD or 10 nM TCDD-10 nM E2, DNase treated, and amplified with primers recognizing the hnRNA, unprocessed RNA, form of CYP1A1 or CYP1A1 mRNA. Results shown are the means and standard deviations of two independent experiments. For MCF-7 and T47D cells, RNA expression levels significantly (P < 0.05) greater than TCDD-treated time matched samples are indicated by an asterisk. For HuH7 samples RNA expression levels significantly (P < 0.05) different between the TCDD-treated time-matched vector control samples and those transfected with ERα are indicated by a asterisk, and RNA expression levels significantly (P < 0.05) different between the TCDD+E2-treated time-matched vector control samples those transfected with ERα are indicated by a number sign.

FIG. 5.

Ligand-dependent recruitment of RIP140 to ERα- but not AhR-regulated target genes. MCF-7 cells were treated with 10 nM estradiol (E2), 10 nM TCDD, 10 nM TCDD-10 nM E2, or DMSO (0.1%) for the indicated amounts of time. ChIP assays were performed with primer pairs specific to the XRE enhancer regions of CYP1A1 or pS2. IgG, immunoglobulin G.

FIG. 6.

ERα plays a role in AhR-mediated activity. Effects of RNAi for ERα on AhR-dependent gene expression was assessed in T47D cells transfected with plasmids containing short hairpin RNA sequences for ERα or luciferase and treated with 10 nM TCDD or 10 nM TCDD-10 nM E2 for 24 h as described in Materials and Methods. RNA was isolated, and the CYP1A1 mRNA levels were determined using real-time PCR. The data are representative of two separate experiments. RNA expression levels significantly (P < 0.05) different between iERα (siRNA for ERα) and the iLuc (siRNA for luciferase) control are indicated by asterisks.

FIG. 7.

Temporal recruitment of ERα to ER and AhR target genes. (A) T47D cells were treated with E2 (10 nM) or TCDD+E2 (10 nM each) for the indicated amounts of time. ChIP assays were performed, followed by PCR amplification of the isolated DNA using primer pairs specific for the enhancer region of CYP1A1 and the promoter of pS2. (B) MCF-7 cells were treated with E2 (10 nM) or TCDD+E2 (10 nM each) for the indicated amounts of time. ChIP assays were performed using anti-ERα antibodies, followed by real-time PCR of the isolated DNA using primer pairs specific for the CYP1A1 and pS2 promoters. The data are representative of an experiment that was repeated twice.

FIG. 8.

New mechanism of cross talk between AhR and ERα. AhR agonists induce the recruitment of ERα to the active AhR, which is enhanced in the presence of E2. Once recruited, ERα can modulate AhR transcriptional activity. Ligand-activated or unliganded ERα can occupy AhR-responsive promoters, reducing the pool of receptors that regulate estrogen-responsive promoters. Recruitment of ERα to the active AhR complex may regulate ERα levels, serving as a mechanism for the proposed AhR-mediated degradation of ERα. E2, estradiol; Ub, ubiquination.