What are comparative studies telling us about the mechanism of ERbeta action in the ERE-dependent E2 signaling pathway?

Abstract

Estrogen hormone (E2) signaling is primarily conveyed by the estrogen receptors (ER) alpha and beta. ERs are encoded by two distinct genes and share varying degrees of domain-specific structural/functional similarities. ERs mediate a complex array of nuclear and non-nuclear events critical for the homeodynamic regulation of various tissue functions. The canonical nuclear signaling involves the interaction of ERalpha and ERbeta with specific DNA sequences, the so-called estrogen responsive elements (EREs). This interaction constitutes the initial step in ERE-dependent signaling in which ERbeta is a weaker transcription factor than ERalpha in response to E2. However, it remains unclear why transactivation potencies of ER subtypes differ. Studies suggest that the amino-terminus, the least conserved structural region, of ERbeta, but not that of ERalpha, impairs the ability of the receptor to bind to ERE independent of E2. Although the impaired ERbeta-ERE interaction contributes, it is not sufficient to explain the weak transactivation potency of the receptor. It appears that the lack of transactivation ability and of the capability of the amino-terminus of ERbeta, as opposed to that of ERalpha, to functionally interact with the carboxyl-terminal hormone-dependent activation domain is also critical for the receptor-specific activity. Thus, the structurally distinct amino-termini of ERs are important determinants in defining the function of ER-subtypes in the ERE-dependent pathway. This could differentially affect the physiology and pathophysiology of E2 signaling.

Figure 1. ERE binding activator and in situ competition assay

(A) Schematics of ERα, ERβ and PPVV, all of which contain an amino-terminus Flag epitope. Two C domains of ERα were co-joined by the D domain to generate the ERE binding module, CDC. The designer transactivator PPVV was engineered by genetically fusing two tandem activation domains of p65 and VP16 to the amino- and carboxyl-termini of CDC, respectively. (B) Comparative transcriptional responses to PPVV and ERs in CHO cells. Cells were co-transfected with 300 ng expression plasmid bearing none (Vector, V), ERα, ERβ or PPVV cDNA and 125 ng the TATA box promoter with one ERE that drives the expression of the firefly luciferase cDNA as a reporter enzyme in the absence or presence of E2 (10⁻⁹) M for 24h. The normalized luciferase activities are presented as fold change compared to the control (V) without E2, which was set to one. The mean ± SEM indicates three independent experiments performed in duplicate. (C) Schematic of the in situ ERE competition assay. (D) The differential effect of E2 on in situ ERE binding of ERα and ERβ in CHO cells. Cells were transfected with 125 ng reporter TATA box promoter bearing one ERE and 300 ng expression plasmid for PPVV, together with varying concentration of expression vector bearing ERα or ERβ cDNA as indicated in the absence or (−E2) or presence (+E2) of 10⁻⁹ M E2 for 24h. The luciferase activity is presented as percentage (%) change compared to control (PPVV alone in the absence of E2, which was set to 100%). The mean ± SEM are three independent experiments performed in duplicate. (E) Chromatin immunoprecipitation (ChIP) assay. CHO cells were transiently transfected with the ERα or ERβ expression vector together with a reporter vector bearing none (TATA) or one ERE (ERE) TATA box promoter. Cells were treated with 10⁻⁷ M E2 for 1h prior to ChIP using a Flag antibody. Sizes of DNA fragments in base-pair are indicated. Shown are modified figures from Huang et al. [46] and used with permission from The Endocrine Society, Copyright 2005.

Figure 2. Structural regions in the amino-terminus of ERβ involved in the impaired receptor-ERE interaction

(A) Schematics of amino-terminal truncations of ERβ. (B) The evaluation of the in situ ERE binding of ERβ variants with the in situ competition assay in CHO cells, which was carried out as described in legend of Fig. 1. Relative ERE binding of receptor species using 300 ng expression vector is depicted as percent (%) decrease in luciferase activity induced by PPVV at 300 ng. (C) The transactivation capacities of ERβ variants in CHO cells. An expression vector bearing none (Vector, V) or an ER variant cDNA was co-transfected with a reporter vector bearing three-tandem consensus EREs upstream of a TATA box promoter driving the expression of the firefly luciferase cDNA (3XERE-TATA-Luc). The mean ± SEM indicates three independent experiments performed in duplicate. Shown are modified figures from Huang et al. [46] and used with permission from The Endocrine Society, Copyright 2005.

Figure 3. Mammalian one- or two-hybrid assays

(A) Schematic of pGal4-TATA-Luc reporter vector, which bear five Gal4 response elements (5xGal4) juxtaposed to the simple TATA box promoter driving the expression of the firefly luciferase cDNA as the reporter. (B) One-hybrid assay was used to assess the intrinsic activation function of the amino-terminus of ERα (amino acids 1–180) or ERβ (amino acids 1–148), which is genetically fused to the Gal4 DNA-binding domain (Gal4). Constructs (300 ng) were transfected into HepG2 cells together with the pGal4-TATA-Luc reporter vector (500 ng). (B) Two-hybrid assay was used to assess the functional interaction between the amino- and carboxyl-termini of ERs. The carboxyl-terminal E/F domain of ERα (amino acids 301–595) or ERβ (amino acids 287–530) was genetically fused to Gal4 DBD, while the amino-terminal A/B region of ERα or ERβ was conjugated to the activation domain (AD) of viral protein 16 (VP16). The proteins were expressed in COS-1 cells together with pGal4-TATA-Luc reporter vector in the presence of 10⁻⁸ M E2 for 48h. The mean ± SEM depicts three independent experiments performed in duplicate. Panel C is a modified figure from Yi et al. [10] and used with permission from The Endocrine Society, Copyright 2005.