Novel DNA motif binding activity observed in vivo with an estrogen receptor α mutant mouse

Abstract

Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the "EAAE" ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.

Figure 1.

Zinc finger domain, DNA motifs, and structural evaluation of the role of E207 in ERE binding selectivity. A, Schematic diagram showing the structure of 2 Zn⁺² fingers of nuclear receptor DBD. Each finger contains 4 cysteine residues (shown in dark red), which coordinately bind a Zn⁺² ion. The first finger contains 3 amino acids that determine specificity for the DNA motif binding (indicated by 2 circles colored green and 1 open circle, the proximal [P] box). The 2 P-box residues colored green were mutated to alanine in the KIKO ERα. The amino acids mutated in the EAAE ERα are colored red. The second finger contains a region involved in ERα dimerization (distal [D] box, open circles). The P-box amino acid residues for ERα (residues 201–216 in the mouse), GR, AR, and PR are shown. The amino acid substitutions used in the KIKO mouse (highlighted in green) and in the EAAE mouse (highlighted in red) are shown. B, Consensus ERE and HRE motif DNA sequences, the preferred motif of KIKO ERα, and NRE, a motif demonstrated to be nonresponsive to nuclear receptors, with palindromic arms numbered, are shown. C, Role of E207 in steric exclusion. Crystal structure of the ERα DBD bound to DNA (blue) (Protein Data Bank code 1HCQ). Modeling of the HRE consensus dT (pink) at position 2 onto the ERE DNA, places the dT C13 methyl group 2.3 Å from E207 of ERα, generating a significant steric clash. In addition, ER-ERE E207 forms a hydrogen bond with the G/C at position 2 of the ERE. Mutation of E207 to A disrupts formation of this hydrogen bond, reducing affinity for the ERE, and also relieves the steric clash with HRE, reducing the selectivity against the HRE.

Figure 2.

Altered hormonal regulation of uterine KLf4 and Klf15. A, RT-PCR of uterine RNA after treatment of WT or KIKO mice for 6 hours with vehicle (V), E₂, or P₄. Statistical analyses included 2-way ANOVA, multiple comparisons of means to results for vehicle treatment, and Bonferroni multiple test correction. ***, P < .001; ****, P < .0001. B, ChIP-seq datasets near Klf15 displayed in University of California Santa Cruz (UCSC) Genome Browser showing WT and KIKO ERα (blue) and PR (red) ChIP-seq tracks from mice treated for 1 hour with vehicle, E₂, or P₄ and input tracks (blue). The arrow shows the HRE motif containing the peak, and the motif sequence that was inserted in pGL4.23 plasmid and tested in the in vitro DNA-binding assay is shown. The HRE motif is indicated by bold text with consensus-matching nucleotides underlined. C, ChIP-PCR for enrichment of ERα at HRE in the Klf15 gene from WT and KIKO uterine samples 1 hour after vehicle or E₂ injection. Statistical analyses included 2-way ANOVA, multiple comparisons of means to results for vehicle treatment, and Bonferroni multiple test correction. **, P < .01; ****, P < .0001.

Figure 3.

KIKO ERα selective computed motif is HRE-like, and in vitro assays demonstrate KIKO binding to the Fkbp5 HRE-motif. A, Called peaks in each dataset (1-hour E₂ WT ERα [blue]: 20 792 peaks, 1-hour E₂ KIKO ERα [yellow]: 18 990 peaks, and 1-hour P₄ WT PR [red]: 12 590 peaks) were compared and are considered overlapping if the coordinate ranges of the peak calls share one or more genomic positions (Table 1). Most overlapping peaks share most their genomic range. Numbers indicate numbers of peaks in each region of the Venn diagram. B, Progesterone response element (PRE) motifs computed from KIKO ERα selective peaks or WT PR ChIP-seq peaks. A total of 12 590 WT P₄ PR peaks were scanned with GADEM analysis seeded with the TRANSFAC PRE motif model (WT PR: 12 590 peaks scanned, 4577 PRE sites [37.8% peaks]; KIKO ERα: 13 385 peaks scanned; 4000 PRE sites [30% of peaks]). C, RT-PCR. Analysis and statistical analysis were performed as described for Figure 2A. D, ChIP-seq datasets near Fkbp5 displayed in the UCSC Genome Browser as described for Figure 2B. E, Luciferase reporter gene activity. Fkbp5 HRE-luc with empty pCDNA3, WT, KIKO, or EAAE ERα or PR, with no hormone (vehicle [V]), E₂ (10 nM), or P₄ (100 nM). Values were calculated relative to those for vehicle treatment of empty expression plasmid and empty reporter plasmid transfected cells. Fold change relative to vehicle treatment is indicated above the bars. Statistical analysis included 2-way ANOVA, multiple comparisons of means to results for vehicle treatment, and Bonferroni multiple test correction. ***, P < .001; **** P < .0001. F, ChIP-PCR. ChIP-PCR analysis and statistical analysis were performed as described for Figure 2C. G, In vitro DNA-binding assay. Biotinylated Fkbp5 HRE binding with nuclear protein extracts from WT, KIKO or EAAE uteri. ERα-DNA complexes were detected as described in Materials and Methods. Nonbiotinylated (unlabeled) DNA (positive control, Fkbp5 HRE; negative control, Fkbp5 NRE) (sequences in Supplemental Table 1) was added to binding reactions at 10× higher levels than the biotinylated probe to compete for ERα binding and demonstrate specificity. Probe, NE sample contained no nuclear extract.

Figure 4.

In vitro assays demonstrate KIKO binding to the Ihh HRE motif. A, RT-PCR. Analysis and statistical analysis were performed as described for Figure 2A. B, Luciferase reporter gene activity. Ihh HRE-luc analysis and statistical analyses were performed as described for Figure 3E. C, ChIP-PCR. ChIP-PCR analysis and statistical analyses were performed as described for Figure 2C. D, In vitro DNA-binding assay. Biotinylated Ihh HRE binding to WT, KIKO, or EAAE ERα-DNA and statistical analysis were performed as described for Figure 3G. Nonbiotinylated (unlabeled) DNA positive control, Ihh HRE; negative control, Fkbp5 NRE.

Figure 5.

In vitro assays demonstrate WT binding to the Igf1 ERE motif. A, RT-PCR. Analysis and statistical analyses were performed as described for Figure 2A. B, Luciferase reporter gene activity. Igf1 ERE-luc analysis and statistical analyses were performed as described for Figure 3E. C, ChIP-PCR. ChIP-PCR analysis and statistical analyses were performed as described for Figure 2C. D, In vitro DNA-binding assay. Biotinylated Igf1 ERE-binding to WT, KIKO, or EAAE ERα-DNA and statistical analysis were performed as described for Figure 3G. Nonbiotinylated (unlabeled) DNA positive control, Igf1 ERE; negative control, Igf1 NRE.

Figure 6.

Evaluation of DBD mutant ERα activities in vivo and in vitro. A, Uterine cross sections from ovariectomized WT, KIKO, EAAE, or ERα-null mice that were treated for 24 hours with E₂. Sections were evaluated for the proliferative marker, Ki67 (brown). Scale bar corresponds to 0.1 mm. B, WT, KIKO, and EAAE ERα mediate AP1-luc responses similarly. WT, KIKO, and EAAE ERα with AP1-luc. Fold changes relative to vehicle (V) treatment are indicated above each bar. E₂, 10 nM; ICI 182,780 (ICI), 100 nM. Statistical analysis included 2-way ANOVA, multiple comparisons of means vs results for vehicle treatment, and Bonferroni multiple test correction. ***, P < .001; ****, P < .0001. C, Microarray profile. A hierarchical cluster of normalized ratios (E₂ 2h/V) of WT and EAAE uterine RNA is shown. Uterine RNA from WT or EAAE ovariectomized mice collected 2 hours after vehicle (V) or E₂ injection (n = 3 each condition) was compared by microarray. The cluster was created by combining replicates and building ratios (E₂ treated/vehicle treated) and filtering for probes that met the following criteria: absolute fold change of > 2 in at least 1 genotype, probe intensity of >100 in at least 1 condition, and false discovery rate < 0.05. This resulted in 3162 probes. Red indicates induced transcripts; green signifies repression.