DNA binding by estrogen receptor-alpha is essential for the transcriptional response to estrogen in the liver and the uterus

Abstract

The majority of the biological effects of estrogens in the reproductive tract are mediated by estrogen receptor (ER)alpha, which regulates transcription by several mechanisms. Because the tissue-specific effects of some ERalpha ligands may be caused by tissue-specific transcriptional mechanisms of ERalpha, we aimed to identify the contribution of DNA recognition to these mechanisms in two clinically important target organs, namely uterus and liver. We used a genetic mouse model that dissects DNA binding-dependent vs. independent transcriptional regulation elicited by ERalpha. The EAAE mutant harbors amino acid exchanges at four positions of the DNA-binding domain (DBD) of ERalpha. This construct was knocked in the ERalpha gene locus to produce ERalpha((EAAE/EAAE)) mice devoid of a functional ERalpha DBD. The phenotype of the ERalpha((EAAE/EAAE)) mice resembles the general loss-of-function phenotype of alphaER knockout mutant mice with hypoplastic uteri, hemorrhagic ovaries, and impaired mammary gland development. In agreement with this phenotype, the expression pattern of the ERalpha((EAAE/EAAE)) mutant mice in liver obtained by genome-wide gene expression profiling supports the observation of a near-complete loss of estrogen-dependent gene regulation in comparison with the wild type. Further gene expression analyses to validate the results of the microarray data were performed by quantitative RT-PCR. The analyses indicate that both gene activation and repression by estrogen-bound ERalpha rely on an intact DBD in vivo.

Fig. 1.

Generation and initial characterization of the ERα^EAAE mutant. The location of the four mutated amino acids in the first zinc finger of the ERα (Y₂₀₁ was changed to E, K₂₁₀ to A, K₂₁₄ to A, R₂₁₅ to E), hence the name EAAE, is indicated (A). In Hela cells, the mutated ERα is unable to activate a luciferase reporter with 2 ERE neither with nor without 10⁻⁸ m E2, but the mutated ERα can still repress a NF-κB-responsive promoter (ICAM-tk-Luc with three NF-κB sites) in an E2-dependent manner (B). Female homozygous ERα^EAAE mice develop hypoplastic uteri (indicated by an arrow) and have blunted mammary gland development (C). In nuclear protein extracts from the livers of wild-type and EAAE mice, the ERα protein is present, but not in liver-specific ERα knockout mice (31 ) (D). Ctrl, Control.

Fig. 2.

Gene expression analysis in the uterus of ERα_(EAAE/EAAE) and wild-type mice. The expression of G(Gdf15) (A), Il17ra (B), Wnt4 (C), Mmd 2 (D), Pgr (E), and p21(F) was examined in the uterus of ERα_(EAAE/EAAE) and wild-type mice (A–F) by qRT-PCR (relative expression level normalized to cyclophilin) V, Vehicle.

Fig. 3.

Igf-1, a uterus-specific ERα target gene, is regulated by direct DNA interaction. The expression of igf-1 was investigated in uterus (A) and liver (B) of wild-type (wt) and ERα_(EAAE/EAAE) mutant (EAAE) mice. The mice were treated with vehicle (V) or EE for 4 or 24 h, and the expression levels of igf-1 determined by qRT-PCR are shown by gray bars described by the left axis (relative expression level normalized to cyclophilin) (A and B). The expression analysis done by GEP is illustrated by black filled squares and described by the right axis (normalized signal intensity) (B).

Fig. 4.

Genome-wide gene expression profiling of RNA isolated from the liver of ERα_(EAAE/EAAE) and wild-type mice and selected single gene RT-PCR analysis. RNA was isolated from the liver of homozygous EAAE mutants and wild-type mice treated for 4 h or 24 h with EE (100 μg/kg) or vehicle, and the cRNA was hybridized to Illumina Mouse SentrixWG-6 version 1.1 Bead Chips. The heat map (hierarchical clustering, Genedata Expressionist) presents the gene expression ratio of EE vs. vehicle (P < 0.001; n = 4–5 per group). The red bars indicate genes that are induced and the green bars indicate genes that are repressed by EE treatment (A). Six genes that, according to GEP with Illumina BeadChips, are induced by EE in the liver of wild-type but not in the EAAE mutant mice (indicated by black squares, left axis) were validated by qRT-PCR (gray bars; right axis): Lifr (B), Il17ra (C), p21 (D), Mmd 2 (E), Tgm2 (F), Gdf15 (G), F3 (H), and Psen2 (I). Mut, Mutant; V, vehicle; wt, wild type.

Fig. 5.

Genes repressed by EE in murine liver. Some of the genes analyzed by the genome-wide expression study are repressed by a 24-h EE treatment. The diagrams show results of the Illumina BeadChip analysis: Gsta4 (A), Arrdc3 (B), Nsbp1 (C), and Ugp2 (D); P values of Welch two-sample t tests describe the significance of gene repression by EE vs. vehicle; V, vehicle.

Fig. 6.

Repression of fnk and LIX in the liver of wild-type, but not ERα_(EAAE/EAAE) mice. To investigate the role of the mutated ERα in repression of gene expression, wild-type and ERα_(EAAE/EAAE) ovariectomized mice were treated sc for 5 d with EE (100 μg/kg) or vehicle (V). On d 5, 1 h after compound treatment, IL-1β (20 μg/kg) was applied by ip injection, which activates NF-κB. The expression level of fnk and LIX was analyzed by qRT-PCR.