The basic helix-loop-helix-PAS protein ARNT functions as a potent coactivator of estrogen receptor-dependent transcription

Abstract

The biological effects of estrogens are mediated by the estrogen receptors ERalpha and ERbeta. These receptors regulate gene expression through binding to DNA enhancer elements and subsequently recruiting factors such as coactivators that modulate their transcriptional activity. Here we show that ARNT (aryl hydrocarbon receptor nuclear translocator), the obligatory heterodimerization partner for the aryl hydrocarbon receptor and hypoxia inducible factor 1alpha, functions as a potent coactivator of ERalpha- and ERbeta- dependent transcription. The coactivating effect of ARNT depends on physical interaction with the ERs and involves the C-terminal domain of ARNT and not the structurally conserved basic helix-loop-helix and PAS (Per-ARNT-Sim) motifs. Moreover, we show that ARNT/ER interaction requires the E2-activated ligand binding domain of ERalpha or ERbeta. These observations, together with the previous role of ARNT as an obligatory partner protein for conditionally regulated basic helix-loop-helix-PAS proteins like the aryl hydrocarbon receptor or hypoxia inducible factor 1alpha, expand the cellular functions of ARNT to include regulation of ERalpha and ERbeta transcriptional activity. ARNT was furthermore recruited to a natural ER target gene promoter in a estrogen-dependent manner, supporting a physiological role for ARNT as an ER coactivator.

Fig. 1.

ARNT interacts with ERα and ERβ and enhances E₂-dependent transcription. (A) COS-7 cells were seeded in 10-cm plates and transfected with 5 μg of pCMV4-ARNT and 5 μg of pSG5-ERα or -ERβ as shown and treated with 10 nM E₂ or 1 μM tamoxifen (T) or vehicle. After transfection, whole-cell extracts were prepared and immunoprecipitation experiments were performed with ERα or ERβ antibodies as described in Materials and Methods. The precipitated material was transferred to nitrocellulose, and the presence of ARNT was monitored by Western blot experiments. (Upper) Precipitated material under different treatments. (Lower) Ten percent input material. (B) HeLa cells were transfected with 10 ng of expression vectors for ERα or ERβ together with 3xERE-TATA-luciferase reporter (500 ng) construct and pCMV5-β-gal (50 ng) reporter gene construct. In selected reactions, increasing amounts of ARNT (100–500 ng) were also introduced as indicated. After transfection, cells were treated with 10 nM E₂ or 1 μM tamoxifen and incubated for 48 h before luciferase measurement. The activity of ERα or ERβ in the presence of E₂ was arbitrarily set to 100%, and the effects of ARNT were compared with this result. Mean of three transfections performed in duplicate is presented. (C) HeLa cells were transfected as above with expression vectors for ERα (Left), ERβ (Right), and ARNT or TIF-2 as indicated together with a 3xERE-luciferase reporter construct. After transfection, cells were treated with 10 nM E₂ or 1 μM tamoxifen, and luciferase activity was measured and plotted.

Fig. 2.

Differential enhancement of ER activity by different ARNT family members. HeLa cells were transfected as described in Fig. 1 with expression vectors for ERα (A) or ERβ (B), an ERE-regulated luciferase reporter gene construct, β-gal internal control, and increasing concentrations of ARNT, ARNT-2, or bMAL. After transfection, cells were treated with 10 nM E₂ for 48 h. After this incubation, cells were harvested and luciferase andβ-gal activity was determined.

Fig. 3.

The C-terminal domain of ARNT is required to enhance ER activity. HeLa cells were transiently transfected with ERα (A and C)or ERβ (B and D) expression vectors, ERE-regulated reporter gene construct, β-gal internal control plasmid, and equal amounts of expression vectors for full-length ARNT or different deletion constructs as shown. Cells were treated and harvested as described in Fig. 1, and luciferase activity was determined and plotted.

Fig. 4.

ARNT interacts with the C-terminal domain of the ERs. (A) HeLa cells were transfected with expression vectors coding for Gal4 fusion proteins of ERα or ERβ together with a Gal4-regulated luciferase reporter construct. After transfection, cells were treated with ligands as in Fig. 1, and the transcriptional response was measured. Luciferase activity from cells transfected with E₂-activated Gal-ERα LBD or Gal-ERβ LBD in the absence of ARNT was arbitrarily set to 100%, and the effect of ARNT was compared with this value. HeLa cells were transfected with expression vectors for ERα (B) or ERβ (C) and corresponding deletion constructs where the N-terminal A/B domain was removed, ERE-regulated luciferase reporter gene construct, β-gal internal control, and increasing concentrations of ARNT. The cells were treated with E₂ for 48 h and harvested, and luciferase activity was measured and corrected against the internal β-gal transfection control. The reporter activity in nontreated cells in the absence of ARNT and ERα (B)or ERβ (C) was arbitrarily set to one for comparison.

Fig. 5.

Time-dependent recruitment of ARNT to the estrogen-responsive pS2 promoter. T47D cells, cultured in the absence of estrogen, were treated without (time 0) and with 10 nM E₂ for 15 and 45 min. Soluble chromatin was prepared and immunoprecipitated by using antibodies raised against rabbit IgG (α-IgG) as negative control, ERα (α-ERα), and two different ARNT antibodies (α-ARNT1). Immunoprecipitated DNA was PCR-amplified with primers that span the -353 to -54 region of the pS2 promoter.