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

ORIGINAL

RESEARCH

Novel DNA Motif Binding Activity Observed In Vivo With an Estrogen Receptor ␣ Mutant Mouse Sylvia C. Hewitt, Leping Li, Sara A. Grimm, Wipawee Winuthayanon, Katherine J. Hamilton, Brianna Pockette, Cory A. Rubel, Lars C. Pedersen, David Fargo, Rainer B. Lanz, Francesco J. DeMayo, Günther Schütz, and Kenneth S. Korach Receptor Biology (S.C.H., W.W., K.J.H., B.P., K.S.K.), Laboratory of Reproductive and Developmental Toxicology, Biostatistics Branch (L.L.), Integrative Bioinformatics (S.A.G., D.F.), Laboratory of Structural Biology (L.C.P.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; Department of Molecular and Cellular Biology (C.A.R., R.B.L., F.J.D.), Baylor College of Medicine, Houston, Texas 77030; and Department of Molecular Biology of the Cell (G.S.), German Cancer Research Center, 69121 Heidelberg, Germany

Estrogen receptor ␣ (ER␣) 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 DNAbinding. 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 ER␣ binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNAbinding assays. Because the KIKO ER␣ has HRE DNA-binding activity, we evaluated the “EAAE” ER␣, 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 ER␣ 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 ER␣ tethering, because KIKO ER␣ effectively stimulates transcription using HRE motifs. The EAAE-ER␣ DNA-binding domain mutant mouse demonstrates that ER␣ DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ER␣ tethering may not contribute to estrogen responsiveness in vivo. (Molecular Endocrinology 28: 899 –911, 2014)

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he estrogen receptor ␣ (ER␣) controls transcriptional rates of target genes in cells by directly interacting with the estrogen response element (ERE) DNA motifs. A second “tethering” mechanism has been described using in vitro studies, in which ER␣ interacts with other DNA motif binding transcription factors, such as FOS/JUN

dimers on AP1 sites (1). To elucidate biological processes mediated by direct ERE vs indirectly tethered responses, mutations were introduced at amino acids 207 and 208 in the second “knuckle” of the first zinc finger of the mouse ER␣, a region referred to as the proximal box (P-box) (Figure 1A), because these residues govern DNA sequence

ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received February 11, 2014. Accepted March 28, 2014. First Published Online April 8, 2014

Abbreviations: ChIP-seq, chromatin immunoprecipitation sequencing; DBD, DNA-binding domain; E2, estrogen; EBNA, Epstein-Barr virus nuclear antigen; ER, estrogen receptor; ERE, estrogen response element; GR, glucocorticoid receptor; HRE, hormone response element; Ihh, Indian hedgehog; NE, nuclear extract; NRE, nonresponsive element; KIKO, heterozygous; KLF, Kruppel-like factor; KIWT, DBD mutation; P4, progesterone; luc, luciferase; Mcm2, minichromosome maintenance 2; P-box, proximal box; PR, progesterone receptor; WT, wild-type; WTKO, ER␣-null allele.

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Mol Endocrinol, June 2014, 28(6):899 –911

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HRE Activity of ER␣ DBD Mutant

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 ER␣. The amino acids mutated in the EAAE ER␣ are colored red. The second finger contains a region involved in ER␣ dimerization (distal [D] box, open circles). The P-box amino acid residues for ER␣ (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 ER␣, 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 ER␣ 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 ER␣, 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.

selectivity (2, 3). When the mutations were knocked in to the mouse ER␣ locus (4), females heterozygous for the ER␣ DNA-binding domain (DBD) mutation (KIWT) were infertile, developed abnormally enlarged uteri, and were anovulatory (4). To overcome these issues and produce a mouse model with the DBD-mutated ER␣ as its only functional ER␣ allele, KIWT males were bred to females heterozygous for the ER␣-null allele (WTKO) (5). The resulting compound heterozygous (KIKO) females were infertile, and although initial findings suggested an ability to mount uterine proliferative responses to estrogen (5), later studies indicated that KIKO uteri are refractory to estrogen response in terms of weight increase and epithelial cell proliferation (6, 7). Further evaluation of the KIKO uterine response revealed lack of estradiol (E2)mediated induction of IGF-1 (Igf1) (7), a growth factor essential for uterine growth (8). A second ER␣ DBD mutant mouse model (EAAE), which has 4 amino acid mutations that flank the P-box region (amino acids 201, 210, 214, and 215) (Figure 1A), also serves as an in vivo model to discern ERE vs tethered responses (9). This second


model differed from the KIKO model in that EAAE mice displayed more of the phenotypes observed in ER␣-null mouse models and lacked female liver transcriptional responses (9). In addition, females heterozygous for the EAAE ER␣ mutations are fertile (9). The activities of ER␣ and progesterone receptor (PR) in uterine tissue are interrelated. Proper uterine function requires intricate regulation of responses to fluctuation in estrogen and progesterone hormone levels, both temporally and in terms of uterine cell type-specific events (10). These effects are balanced, with the PR-mediated-P4 function tempering the pro-proliferative activity of the ER␣mediated E2 response. Two Kruppel-like factors (KLFs) have been implicated in E2 and P4 modulation of uterine proliferation; KLF4 is increased by E2 and promotes DNA replication, whereas KLF15 is increased by P4 and inhibits growth via regulation of minichromosome maintenance 2 (Mcm2) (11). We hypothesized that tethered ER␣ signaling mediated by the DBD mutant was insufficient to regulate uterine biological functions. Therefore, we evaluated the regulation of Klf4 and Klf15 to determine whether their mis-regulation might contribute to the inability of E2 to induce uterine growth. Our study has revealed not only an inability of KIKO ER␣ to induce uterine Klf4 but also an unexpected E2 induction of the normally P4-responsive Klf15 by KIKO ER␣. Recently, we described the uterine ER␣ “cistrome,” showing sites of interaction between ER␣ and chromatin in mouse uterine tissue, by using chromatin immunoprecipitation sequencing (ChIP-seq) (12). Similarly, Rubel et al (13) have described the uterine PR cistrome. Therefore, to evaluate the mechanisms that might underlie the E2 induction of Klf15 by KIKO ER␣, we examined the KIKO ER␣ cistrome to determine sites of interaction of KIKO ER with uterine chromatin, expecting to determine tethered sites but have instead revealed altered DNA motif interaction of the KIKO ER␣.

Materials and Methods Animals and tissue samples All procedures with animals were performed under an animal study protocol approved by the National Institute of Environmental Health Sciences Animal Care and Use Committee in accordance with policies detailed in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult female wild-type (WT) (C57B/6), KIWT (B6;129P2-Esr1⬍tm1Lja⬎), EAAE (B6; 129P2-Esr1⬍tm2.1Gsc⬎, WTKO (B6.129-Esr1⬍tm4.2Ksk⬎), and KIKO (B6;129-Esr1⬍tm4.2Ksk⬎Esr1⬍tm1Lja⬎) mice were produced in our breeding colony at Charles River Laboratories as described previously (6) and shipped to National Institute of Environmental Health Sciences. For the KIWT uterine enlargement study, tissue from 7- to 18-week-old female mice was collected and


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weighed. For hormone response studies, adult female mice were ovariectomized and then were rested for 10 to 14 days to allow endogenous ovarian hormones to clear. WTKO female mice were used as controls for the KIKO mice because they express ER␣ from 1 allele, as do the KIKO mice, and are called “WT” in the text to simplify the descriptions. Animals were treated as indicated, and uterine tissue was collected and snap-frozen in liquid nitrogen for RNA, protein, or chromatin isolation. RNA isolation and RTPCR, microarray, and ChIP-PCR were performed as described previously, including most primer sequences (6, 12, 14) with the addition of those listed in Supplemental Table 1. Microarray data were deposited at Gene Expression Omnibus (GSE56423). For ChIP-PCR, a primer set was selected in the aquaporin 5 (Aqp5) transcript region with no ER enrichment as a negative control. For Western blotting, whole-tissue homogenates were made as described previously (7), and 10 ␮g of protein was loaded on each lane of a 10% NuPAGE gel (Invitrogen). Proteins were transferred to nitrocellulose using iBlot (Invitrogen) and probed for ER␣ (1: 1000, sc-542; Santa Cruz Biotechnology) and ␤-tubulin (1:5000, sc-9104; Santa Cruz Biotechnology) in 5% Blotto (Santa Cruz Biotechnology) in Tris-buffered saline-Tween 20 buffer and detected using infrared fluorescence–labeled (800 nm) antirabbit antibody (1:20 000; LI-COR) and scanned and quantified using an Odyssey Fc instrument and Image Studio software (LI-COR).

ChIP-seq For ER␣ ChIP-seq, E2 (250 ng in 100 ␮L of saline; Research Plus) or 100 ␮L of saline was injected intraperitoneally in each of 5 mice/treatment group. One hour after injection, mice were killed. Then uterine tissue was collected, snap-frozen in liquid nitrogen, pooled, and shipped to Active Motif for FactorPath ChIP-seq. Each dataset was obtained from a single pool of uterine tissue (n ⫽ 5 mice). Details of the ChIP-seq using anti-ER␣ antibody (sc-542; Santa Cruz Biotechnology) and alignment to the reference genome were as described previously (12). Mapped reads were deduplicated using MarkDuplicates.jar from the Picard tools package (v1.62; http://picard.sourceforge.net). The Partek Genomics Suite (v6.11.1101; Partek, Inc) was used to make peak calls for ER␣ with default parameters, followed by further filtering to exclude peaks with a Mann-Whitney P value of ⬎.01. Partek Genomics Suite was also used for empirical estimation of the average fragment size per ChIP-seq library. To enable comparison to PR, the PR ChIP-seq dataset (13, GEO GSE 34927) was reanalyzed using the same deduplication, Partek peak calling, and peak filtering steps described above. Characteristics of the ER and PR ChIPseq data are summarized in Supplemental Table 2. Data were deposited in Gene Expression Omnibus (WT ER GSE36455, KIKO ER GSE56466).

Motif enrichment analysis Sequences of peaks unique to WT or KIKO ER␣ or peaks in common were normalized to 300 nucleotides, evaluated with the GADEM tool (15) for motifs, and then compared for relative enrichment.

Reporter assays Candidate motifs identified in ChIP-seq peaks were synthesized as oligonucleotides (Sigma Genosys) (sequences in Supplemental Table 1), annealed, phosphorylated with T4 polynucleotide kinase (New England Biolabs) and then inserted into the


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EcoRV site of the pGL4.23 luciferase (luc) vector (Promega), which is engineered for minimal basal activity, to test candidate response elements. Clones were sequenced to verify the presence of inserted response elements, and clones containing 2 copies of Fkbp5 hormone response element (HRE), Fkbp5 nonresponsive element (NRE), Indian hedgehog (Ihh) HRE, or Epstein-Barr virus nuclear antigen (EBNA) or 3 copies of Igf1 ERE or Igf1 NRE were used in experiments. NRE was made by changing the nucleotides in the Fkbp5 HRE and Igf1 ERE motifs (Supplemental Table 1 and Figure 1B) to match an NRE motif with a motif sequence intermediate between ERE and HRE, which was previously shown to lack activity through ER or glucocorticoid receptor (GR) (16, 17). EAAE ER␣ was made by amplifying the SmaI fragment of mouse ER␣ cDNA in pGEM-3Zf with sitedirected mutagenesis primers (Supplemental Table 1) to recreate the originally described point mutations (Figure 1A) (9). The NotI-XbaI fragment of mouse ER␣ containing the EAAE mutations was used to replace the same fragment of the WT mouse ER␣ cDNA in pCDNA3. The pAP1-luc plasmid was purchased from Stratagene. HeLa cells were transfected as described previously (18) with 0.1 ␮g of reporter constructs and 0.1 ␮g of pCDNA3 expression vectors containing WT, KIKO, or EAAE mouse ER␣ or human PRB, and 0.1 ␮g of pRL-TK control vector. After hormone treatment (0.1% ethanol [vehicle], 10 nM E2, 100 nM P4 [Sigma-Aldrich], or 100 nM ICI 182,780 [Tocris Bioscience]), cell lysates were assayed for luc as described previously (18) and normalized to a Renilla luc vector transfection control. Values were plotted relative to pCDNA3 expression vector plus empty PGL4.23 luc vector transfection with vehicle treatment, except for the AP1-luc assay, which was plotted relative to empty pCDNA3 plus AP1-luc with vehicle treatment.

DNA-binding assay Ovariectomized WT, KIKO, or EAAE mice were injected intraperitoneally with 250 ng of E2 in saline vehicle, and uterine tissue was collected and snap-frozen in liquid nitrogen 1 hour after injections. Pools of 3 to 5 frozen uteri were pulverized, and nuclear extracts (NEs) were prepared (N-PER Kit; Thermo Scientific), as described in the manufacturer’s protocol by homogenization in 200 ␮L of CERI buffer. The ER␣ level in each NE was evaluated using Western blotting, as described above so that the amounts of ER␣ protein used in the binding assays of WT, KIKO, or EAAE samples were the same. NEs were used with the NoShift DNA-binding assay kit according to the protocol provided (Millipore Corp). Biotinylated ERE or HRE probes (Sigma Genosys) (sequences in Supplemental Table 1) were mixed with 2 ␮L of NEs in the presence of 150 nM E2 and P4 and allowed to bind for 60 minutes on ice. Binding reactions were transferred to avidin-coated wells and incubated at 37°C for 1 hour. Bound ER-biotinylated DNA complexes were detected with anti ER␣ antibody (1:300) followed by horseradish peroxidase-labeled antirabbit IgG (Cell Signaling Technology), developed with tetramethylbenzidine substrate, and stopped with 1 N HCl. Wells were scanned to read A450 (SpectraMax Plus 384; Molecular Devices). Specificity of binding was evaluated by including 10- to 20-fold excess unbiotinylated ERE or HRE DNA in the binding step to inhibit complex formation. In addition, excess unbiotinylated NRE, made by altering ERE or HRE, or EBNA, a motif for a different type of transcription


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factor, was added to some binding reactions because these should not interfere with complex formation.

Statistical analyses For RT-PCR, luc reporter activity assays, and ChIP-PCR, data (means ⫾ SEM) were analyzed using the statistical tools in GraphPad Prism 6. Treated groups within each genotype were compared with the vehicle group using 2-way ANOVA; further details are found in the figure legends.

Results E2 induces DNA synthesis inhibitor Klf15 in KIKO uterus Recently, 2 KLFs were implicated in E2 and P4 modulation of uterine proliferation; KLF4 is increased by E2 and promotes DNA replication, whereas KLF15 is increased by P4 and inhibits growth via regulation of


Mcm2, which is important for the initiation of DNA synthesis (11). Because uterine epithelial proliferation is absent in the KIKO uterus, we reasoned that tethered ER␣ signaling might be insufficient to establish proper regulation of these KLFs; therefore, we evaluated their transcripts. E2 robustly increased the Klf4 transcript in WT uteri, but the increase was minimal in KIKO uteri (Figure 2A). As was previously reported in WT mice (11), the Klf15 transcript was increased by P4; however, KIKO uteri exhibit E2-mediated induction of Klf15 (Figure 2A), consistent with the lack of a KIKO uterine proliferative response. Enriched transcription factor motifs within ER␣ ChIP-Seq peak DNA sequences suggests KIKO DNA-binding mutant binds HRE motifs To evaluate the mechanism by which the DBD mutant KIKO ER␣ regulates this normally P4-responsive tran-

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 ER␣ (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 ER␣ 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.


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script, KIKO ER␣ binding to uterine chromatin was evaluated by ChIP-seq and compared with our previously reported datasets from WT uteri. A total of 5605 (30%) of 18 990 KIKO E2 peaks overlapped with WT E2 peaks (Table 1 and Figure 3A). These findings are consistent with the unique E2-induced transcriptional responses of the KIKO uterus (6). To discover transcription factors involved in tethering the KIKO ER␣ to uterine chromatin, WT and KIKO ER␣ ChIP-seq datasets were evaluated for DNA-binding protein motif enrichment using the GADEM tool (15). For this analysis, the WT and KIKO 1-hour E2 peaks were divided into three categories: those unique to the WT set (15 163 WT selective, DNA-binding), those unique to the KIKO set (13 385 KIKO selective), and those common between WT and KIKO (5605 overlapping, presumably tethered ER binding; referred to as WT⫹KIKO overlap hereafter). WT selective, KIKO selective, or WT⫹KIKO overlap sequences were then evaluated for transcription factor– binding motifs and motif enrichment relative to one another. As anticipated, GGTCA/ERE motifs were significantly enriched in WT selective peaks in comparison to the WT⫹KIKO overlap (Supplemental Table 3). Similarly, motifs for numerous known mediators of tethered ER␣ interaction, including Sp1 and AP2, were enriched in WT⫹KIKO overlap vs WT selective peaks (Supplemental Table 4). The enriched motifs represent potential tethering mediators used in vivo by ER␣ in uterine tissue. Remarkably, analysis of motifs enriched at sites of KIKO ER␣ binding revealed that androgen receptor (AR), PR, and GR motifs (androgen response element, progesterone response element, and glucocorticoid response element) dominate those enriched in the KIKO selective vs WT⫹KIKO overlapping peaks (Table 2). The DNA motifs for these 3 receptors Table 1.

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share homology and are often called HREs. To evaluate the possibility that KIKO ER␣ was enriched at PR-binding sites, mouse uterine ChIP-seq profiles from WT PR (13), KIKO ER␣, and WT ER␣ were compared (Figure 3A and Table 1). A total of 2095 (17%) WT PR peaks colocalize with WT ER␣ peaks, whereas 4051 (32%) WT PR peaks overlap with KIKO ER␣ binding peaks (Table 1 and Figure 3A), indicating more overlap between WT PR and KIKO ER␣ than between WT PR and WT ER␣. Based on these observations, we hypothesized that the KIKO ER␣ DBD mutation altered DNA motif specificity. Supporting this hypothesis, the HRE binding motif computed from the PR binding peaks is similar to one computed from the KIKO selective peaks (Figure 3B). To explore whether KIKO ER␣ induces the Klf15 transcript via an HRE motif, the ChIP-seq data were examined and revealed PR and KIKO ER␣ enrichment in the second intron that contains a consensus HRE (Figure 2B). ChIPPCR confirmed E2-dependent enrichment of KIKO ER␣ at this HRE (Figure 2C). In vitro HRE reporter and DNA-binding assays demonstrate interaction between KIKO ER␣ and HRE motifs To further test our hypothesis that the KIKO ER␣ can bind HRE motifs, we developed in vitro assays by evaluating typically P4-responsive transcripts that are induced by E2 in KIKO uteri. FK506 binding protein 5 (Fkbp5) is a large-molecular-weight immunophilin and is a wellknown target of PR or GR (19). RT-PCR analysis of uterine RNA shows that Fkbp5 is significantly increased by P4 in the WT uterus (Figure 3C). However, in the KIKO uterus, the transcript is robustly increased by E2. Examination of the WT and KIKO ER␣ and PR ChIP-seq

Comparison of WT ER␣, KIKO ER␣. and PR ChIP-seq called peaks No of Peaks in Sample Y (%)

No. of Sample Peaks in “X” Sample X 1-h V WT ER␣ 5496 1-h E2 WT ER␣ 20792 1-h V KIKO ER␣ 2908 1-h E2 KIKO ER␣ 18990 1-h V WT PR 3541 1-h P4 WT PR 12590 1-h E2 WT ER␣ selective 15163 1-h E2 KIKOER␣ selective 13385 1-h E2 WT ER␣ 5605 ⫹1-h E2 KIKO ER␣

1-h V WT ER␣ ⫺ 3968 (19.1) 1124 (38.7) 2580 (13.6) 1169 (33.0) 1136 (9.0) ⫺ ⫺ ⫺

1-h E2 WT ER␣ 3992 (72.6) ⫺ 1360 (46.8) 5605 (29.5) 1558 (44.0) 2095 (16.6) ⫺ ⫺ ⫺

1-h V KIKO ER␣ 1125 (20.5) 1352 (6.5) ⫺ 2025 (10.7) 720 (20.3) 835 (6.6) ⫺ ⫺ ⫺

1-h E2 KIKO ER␣ 2596 (47.2) 5629 (27.1) 2037 (70) ⫺ 1947 (55.0) 4051 (32.2) ⫺ ⫺ ⫺

1-h V WT PR 1169 (21.3) 1549 (7.4) 723 (24.9) 1938 (10.2) ⫺ 2132 (16.9) ⫺ ⫺ ⫺

1-h P4 WT PR 1133 (20.6) 2090 (10.1) 838 (28.8) 4021 (21.2) 2133 (60.2) ⫺ 586 (4.0) 2468 (18.0) 1388 (25.0)

Comparing WT ER␣, KIKO ER␣, and WT PR ChIP-seq peaks reveals KIKO ER␣ selective enriched sites and overlap with PR enriched regions. Peaks (% of total peaks) from different samples that share one or more genomic bases in common are considered overlapping peaks. Of the peaks called in sample X, how many (what % of X) overlap with a peak called in sample Y? Note that the reciprocal comparisons should be very similar, but perhaps not identical because 1 peak from sample X might overlap with 2 peaks in sample Y, which is counted as 1 for X vs Y, but as 2 for Y vs X. Most overlapping peaks share the majority of their genomic range. One-hour E2 WT or KIKO ER␣ selective peaks are those that do not overlap with each other (total peaks minus overlapping peaks). Comparisons in bold type are discussed in the text.


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Figure 3. KIKO ER␣ 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 ER␣ [blue]: 20 792 peaks, 1-hour E2 KIKO ER␣ [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 ER␣ 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 ER␣: 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 ER␣ 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. ER␣-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 ER␣ binding and demonstrate specificity. Probe, NE sample contained no nuclear extract.


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tified in the sequence within this peak (Supplemental Table 1) and inserted into a pGL4.23 luc reporter vector to evaluate the ability of WT, ER␣, both KIKO and EAAE ER␣ (DBD mutants) and PR to bind to the HRE and Model Enrichment mediate hormone-dependent transcription of the luc reName P Value porter gene. We reasoned that an ability of KIKO ER␣ to AR_01 1.34E–105 bind HRE motifs would be reflected in an estrogen-depenAR_02 1.35E–100 AR_04 1.23E–95 dent increase in luc activity. WT ER␣, KIKO ER␣, and PR AR_03 3.21E– 67 all induced hormone-dependent luc activity, whereas EAAE GR_Q6 3.47E–55 ER␣ was inactive (Figure 3E). ER␣ ChIP-PCR revealed E2PR_02 1.52E–52 GR_01 1.10E–39 dependent recruitment of KIKO but not WT ER␣ to this site PR_01 2.73E–33 in uterine tissue (Figure 3F). An in vitro DNA-binding assay AR_Q2 2.37E–23 revealed KIKO ER␣ but not WT or EAAE ER␣ interaction GR_Q6_01 5.15E–16 with this Fkbp5 HRE (Figure 3G). PR_Q2 9.96E–10 GRE_C 8.67E– 09 Ihh is a member of the hedgehog signaling family, and AREB6_02 2.14E– 08 its proper regulation is essential to establish pregnancy HSF1_01 7.39E– 08 (10). Ihh is increased in WT uteri after P4 injection (Figure SOX4_01 1.13E– 07 SOX9_B1 1.86E– 07 4A). This transcript is increased by P4 or E2 in the KIKO DELTAEF1_01 6.20E– 07 uterus. Peaks of hormone-dependent PR or KIKO ER␣ AREB6_01 9.95E– 07 binding enrichment are seen 5⬘ of Ihh, in an intron of Abbreviations: ARE, androgen response element; GRE, glucocorticoid Nhej1, a region that was previously described as a potenresponse element; PRE, progesterone response element. tial mediator of PR induction of Ihh (20) (Supplemental profiles near the Fkbp5 transcript revealed a region lo- Figure 1A). Some WT ER␣ recruitment also occurs in this cated in an intron with E2-dependent WT ER␣ recruit- region. An HRE motif was identified in the sequences in ment, P4-independent PR binding, and E2-dependent the peak (Supplemental Table 1). This motif mediated KIKO ER␣-binding (Figure 3D). An HRE motif was idenE2-dependent KIKO ER␣ luc activity or P4-dependent PR activity (Figure 4B), reflecting interaction with the HRE sequence. WT and EAAE ER␣ were unable to induce the Ihh HRE luc reporter. ChIP-PCR confirmed recruitment of KIKO ER␣ to this site in uterine tissue (Figure 4C). An in vitro DNA-binding assay revealed KIKO ER␣ but not WT or EAAE ER␣ interaction with this Ihh HRE (Figure 4D). Our previous analysis demonstrated that the Igf1 transcript is increased by E2 in the WT uterus but not in the KIKO uterus (Figure 5A) (7). In addition, using ChIP-seq we have identified 5 sites of ER␣ enrichment 50 kb 5⬘ of the Igf1 transcription start site (12). Some KIKO ER␣ enrichment occurs at 2 of the 5 sites in this region (Supplemental Figure Figure 4. In vitro assays demonstrate KIKO binding to the Ihh HRE motif. A, RT-PCR. Analysis 1B). To demonstrate the validity of 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. our in vitro assay analysis strategy, C, ChIP-PCR. ChIP-PCR analysis and statistical analyses were performed as described for Figure we tested an ERE motif that was 2C. D, In vitro DNA-binding assay. Biotinylated Ihh HRE binding to WT, KIKO, or EAAE ER␣-DNA identified in one of the sites that and statistical analysis were performed as described for Figure 3G. Nonbiotinylated (unlabeled) DNA positive control, Ihh HRE; negative control, Fkbp5 NRE. lacked KIKO ER␣ enrichment (Sup-

Table 2. HRE Motifs (ARE, GRE, and PRE) Dominate Enriched Motifs in 1-Hour E2 KIKO ER␣ Selective vs 1-Hour E2 WT ER␣ ⫹ 1-Hour E2 KIKO ER␣ Overlapping Peaks


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mutation are fertile (9), indicating a difference in the biological functions mediated by these two DBD mutant estrogen receptors. Unlike the KIKO ER␣, the EAAE ER␣ completely lacks binding to both ERE and HRE motifs (Figures 3G, 4D, and 5D) and is unable to induce ERE-luc or HREluc reporter constructs (Figures 3E, 4B, and 5B and Supplemental Figure 2). EAAE ER␣ induces an AP1-luc reporter comparably with WT or KIKO ER␣ (Figure 6B). To examine potentially tethered functions mediated by EAAE ER␣ in uterine tissue, we evaluated genes regulated by E2 after 2 hours by microarray and RTPCR (Figure 6C and Supplemental Figure 4A). Although the EAAE 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 ER␣ protein is expressed in the tisactivity. Igf1 ERE-luc analysis and statistical analyses were performed as described for Figure 3E. sue (Supplemental Figure 4B), most C, ChIP-PCR. ChIP-PCR analysis and statistical analyses were performed as described for Figure of the transcriptional responses are 2C. D, In vitro DNA-binding assay. Biotinylated Igf1 ERE-binding to WT, KIKO, or EAAE ER␣-DNA absent (Figure 6C and Supplemental and statistical analysis were performed as described for Figure 3G. Nonbiotinylated (unlabeled) DNA positive control, Igf1 ERE; negative control, Igf1 NRE. Figure 4C), including both induced and repressed transcripts. This result demonstrates that ER␣ DNA-binding plemental Table 1). WT ER␣ robustly induced this EREluc reporter, but KIKO ER␣ induced minimal activity, is required for both up- and down-regulation of transcripand EAAE ER␣ was inactive (Figure 5B). ChIP-PCR re- tion in the uterus. Of the 3163 probes shown clustered in vealed selective recruitment of WT ER␣ to this site in Figure 6C, only 53 probes representing 44 genes (1.6%) uterine tissue (Figure 5C). Although KIKO ER␣ showed were E2 regulated in the EAAE uteri (⬎2-fold change, false little Igf1 ERE-luc reporter activity, the in vitro DNA- discovery rate ⬍ 0.05) (Supplemental Figure 4C). Of the 53 binding assay revealed both WT ER␣ and KIKO ER␣ differentially expressed EAAE transcripts, 68% were ininteraction with this ERE (Figure 5D), whereas EAAE creased, 32% were decreased, and, overall, only 13 of the 53 ER␣ lacked interaction. KIKO ER␣ binding to the ERE is probes were regulated in the same direction in WT and also apparent in the increased basal ERE-luc activity com- EAAE uteri, whereas 29 probes were regulated in opposite pared with that for EBNA motif-luc (compare Figure 5B directions, and 13 were not differentially expressed in the and Supplemental Figure 2), a motif that does not bind WT. Significantly, transcripts that were previously observed KIKO ER␣, as including 10-fold excess unlabeled EBNA as E2 responsive in the KIKO uterus (6) are not responsive in motif DNA does not prevent KIKO ER␣ binding to any of the EAAE uterus (Supplemental Figure 4, A and D). Overall, the biotinylated probes (Supplemental Figure 3). these in vitro and in vivo findings suggest that ER␣ tethering in uterine tissue is not sufficient to mediate hormonal rePhenotypic differences between KIKO and EAAE sponsiveness including most transcriptional responses, beDNA-binding-deficient mouse models cause the EAAE mouse model lacks uterine estrogen The EAAE ER␣ DBD mutant mouse has phenotypes responses. similar to those of ER␣-null animals (9). Although KIKO uteri are similar in size to WT uteri, the EAAE uteri are hypoplastic, similar to ER␣-null uteri (9) (Figure 6A); Discussion however, neither DBD model exhibits uterine epithelial proliferation (Figure 6A). Female mice that are heterozy- Altered DNA-binding specificity rather than DNAgous for the KIKO allele (WTKI) develop enlarged uterine binding deficiency Nuclear receptors can be categorized according to tissue after puberty (Supplemental Table 5) and are infertile (4); however, females heterozygous for the EAAE ER␣ their preferences for DNA motif sequence binding. ER,


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places the dT C7 methyl group 2.3 Å from the side chain of E207, generating a significant clash (Figure 1C). Removal of the side chain for E207, as in the KIKO ER␣ E207A mutation, would remove this steric clash with HRE or NRE motifs. Note that the preferred motif for KIKO ER␣ that we determined in this study indicates relaxed preference at position 2 of the motif (Figures 1B and 3B). In contrast, when the structure of the EAAE mutant ER␣ is examined, an H bond normally formed between K210 of WT ER␣ and G/C at position 1 in the ERE is lost; thus, interaction with ERE is not favored. Moreover, interaction between the EAAE ER␣ and GAACA HRE moFigure 6. Evaluation of DBD mutant ER␣ activities in vivo and in vitro. A, Uterine cross sections from ovariectomized WT, KIKO, EAAE, or ER␣-null mice that were treated for 24 hours with E2. tifs is excluded, because this mutant Sections were evaluated for the proliferative marker, Ki67 (brown). Scale bar corresponds to 0.1 retains E207, maintaining the steric mm. B, WT, KIKO, and EAAE ER␣ mediate AP1-luc responses similarly. WT, KIKO, and EAAE ER␣ conflict with A:T at position 2. 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 Therefore, these modeling observaof means vs results for vehicle treatment, and Bonferroni multiple test correction. ***, P ⬍ .001; tions suggest that the KIKO ER␣ in****, P ⬍ .0001. C, Microarray profile. A hierarchical cluster of normalized ratios (E2 2h/V) of teraction with HRE is possible, but WT and EAAE uterine RNA is shown. Uterine RNA from WT or EAAE ovariectomized mice that interaction between EAAE ER␣ 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 and HRE is not. In concordance treated/vehicle treated) and filtering for probes that met the following criteria: absolute fold with these observations, mutation of change of ⬎ 2 in at least 1 genotype, probe intensity of ⬎100 in at least 1 condition, and false the human ER␣ K210 (equivalent to discovery rate ⬍ 0.05. This resulted in 3162 probes. Red indicates induced transcripts; green signifies repression. mouse K214) decreases ERE binding to a degree similar to that of muvitamin D receptor and thyroid receptor prefer motifs tating human ER␣ E203A (equivalent to mouse E207), with GGTCA arms, whereas AR, GR, and PR prefer but does not lead to interaction with A:T at position 2 GAACA (21, 22). Analysis of the sequences in KIKO ER␣ (23). In addition, a single mutation of human ER␣ E203 selective peaks revealed a preference for motifs with the to A remained permissive for interaction with ERE (23), GAACA sequence, normally associated with AR, PR, or consistent with the KIKO ER␣ ERE binding observed in GR binding. The mutations in KIKO ER␣ (E207A and the DNA-binding assay (Figures 5D and Supplemental G208A) are located at the base of the first zinc finger Figure 3). Similar to what we have reported using the (Figure 1A). Glutamate and glycine (EG) are found in this KIKO mouse uterus (6), transcriptional profiling of E2P-box region of all other nuclear receptors that bind the treated cells with ER␣ P-box mutations that “switch” the GGTCA motif, including thyroid receptor and vitamin D EG to GS revealed regulation of transcripts that were receptor (2, 22). The amino acids in the “knuckle” of the unique to the DBD mutant ER␣ (24), and the ER␣ ChIPfirst zinc finger in the P-box of nuclear receptors that bind seq revealed ER␣-binding sites that were unique to the to the GAACA motif (GR, PR, and AR) are glycine and DBD mutant ER␣ and enriched for HRE motifs (24). serine (GS) (22). Examination of crystal structures of Recently, a role for tethering through AP1 has been DBDs of ER␣, PR, and GR bound to their cognate DNA proposed in an assisted loading mechanism whereby acmotifs (ERE, PRE, and GRE) as a basis to predict the tivation of GR leads to increased chromatin accessibility effect of the amino acid substitutions in the KIKO ER␣ for ER␣ at a small fraction of ER␣-binding sites in mamsuggests that E207 may contribute to the specificity of mary epithelial cells (25). The KIKO DBD mutant ER␣ ER␣ for the ERE over HRE sequences by steric exclusion was used to demonstrate that ERE-binding activity was of the dT:dA at position 2 nucleotide present in the HRE not needed for the assisted loading (25). The sites that (Figure 1C). Modeling of a dT:dA at ERE position 2 mediate the assisted loading were shown to be enriched


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for the HRE motif (25); thus, the HRE binding of the KIKO DBD mutant ER␣ that we demonstrate in our study may be directing the binding. AP1 binding is enriched and required at these assisted loading regions (25), but its role in the mechanism might need to be further clarified. KIKO ER␣ DNA-binding motif specificity Our in vitro DNA-binding analysis (Figures 3G, 4D, and 5D and Supplemental Figure 3) indicated that the KIKO ER␣ bound both ERE and HRE sequences, and, in addition, the negative control unlabeled competitor (NRE; Supplemental Table 1 and Figure 1B) was effective in competing for interaction with KIKO ER␣. The NRE, with a sequence intermediate between ERE and HRE (Figure 1B), was used based on work indicating that this motif did not bind ER␣ (17) or mediate ER or GR transcription (16). Therefore, the ability of the NRE to inhibit KIKO ER␣ binding to the ERE or HRE suggests that the DNA motif preference of KIKO ER␣ is quite relaxed and includes sequences that are typically unable to bind steroid receptors. Specificity was demonstrated using the unrelated EBNA motif, which did not bind KIKO ER␣ (Supplemental Figure 3). In the reporter assay study (Figures 3E, 4B, and 5B and Supplemental Figure 2), KIKO ER␣ was active via HRE-luc and NRE-luc but not EREluc or EBNA-luc reporters. However, KIKO ER␣ increased basal activity of the Igf1 ERE-luc reporter (Figure 5B), suggesting that some interaction is occurring, but that E2 is unable to induce transcription via the KIKO ER␣ on the ERE. Overall, by comparing the motifs used to test binding to KIKO ER␣ and its ability to induce luc reporter activity, the common nucleotides indicate a “consensus” motif of GnnCAnnnTGnnC (Figure 1B and Supplemental Table 6). WT ER␣ showed a strict preference for ERE in the in vitro DNA-binding assay (Figure 5D and Supplemental Table 6); nonetheless, WT ER␣ mediated E2-dependent activity via the Fkbp5 HRE-luc (Figure 3E) and the Fkbp5 NRE-luc (Supplemental Figure 2) but not the Ihh HREluc (Figure 4B). The activity may be driven by DNA sequences flanking the Fkbp5 HRE and NRE, because comparable WT ER␣ transcriptional activity is seen with Fkbp5 HRE-luc (Figure 3B), and Fkbp5 NRE-luc (Supplemental Figure 2), but none occurs via Igf1 NRE-luc (Supplemental Figure 2). Although there were indications in these in vitro assays for a WT ER␣ interaction with Fkbp5 HRE-luc, this interaction was not apparent in the conditions found in the uterine tissue, because WT ER␣ was not enriched by ChIP-PCR (Figure 3F). The HRE motif tested in the Fkbp5 construct was previously iden-


tified and similarly demonstrated by reporter assay and gel shift to interact with PR and GR (19). Potential biological significance AR, GR, and PR are all present in the mouse uterus, but the biological role of PR is most closely linked to that of ER␣, allowing responses to cycling ovarian hormones, E2 and P4. ER␣ and PR exhibit a well-characterized balance in activities in terms of cycle stage (periovulatory vs early pregnancy) and uterine tissue compartments (stroma vs epithelia). PR is restricted to the epithelia until the preovulatory increase in E2 leads to decreased epithelial and increased stromal expression of PR. ER␣ is found in all cell types of the uterine tissue, and proper levels and patterns of PR help to maintain an appropriate estrogen response (10). We speculate that rather than a model for tethered-selective ER␣ signaling, the KIKO mouse exhibits estrogen regulation of target genes normally controlled by PR and inappropriate induction of PR target genes in the stromal compartment. Perhaps this underlies the inability of E2 to cause KIKO uterine proliferation (6, 7), because PR normally inhibits epithelial proliferation to allow embryo attachment and implantation (26). As an example, Ihh, which has been shown to be involved in the PR-mediated inhibition of epithelial proliferation necessary for embryo implantation (27), becomes E2 inducible in the KIKO uterus. The uterine enlargement reported for the KIWT heterozygotes (Supplemental Table 5 and Ref. 4) is similar to observed uterine overgrowth in a model with constitutive activation of the IHH transducer, smoothened (SmoM2⫹) (28). We hypothesize that estrogen might be increasing IHH via KIKO ER␣ HRE interaction in a setting that maintains ER␣ ERE-mediated responses through the WT allele. Consistent with our hypothesis, KIWT uterine enlargement develops with the onset of puberty (Supplemental Table 5). When estrogen elevates Ihh/SMO activation in the KIKO uterus, ER␣ signaling via ERE is impaired. Conversely, when estrogen elevates Ihh in KIWT uterine tissue, resulting in SMO activation, in a milieu where WT ER␣ signaling is intact, enlargement develops. This difference emphasizes an important role for ERE-mediated responses in the overgrowth. Similarly, normally P4, but not E2, increases epithelial KLF15, which then inhibits expression of Mcm2, thereby preventing epithelial proliferation (11). However, KIKO ER␣ binds a chromatin site within the Klf15 transcript that contains a consensus HRE, and Klf15 is increased by E2 (Figure 2), which may prevent the KIKO uterine proliferation. More severe mutation of the DBD was used in the EAAE ER␣ and leads to an ER␣ null-like female reproductive tract phenotype and lack of estrogen induced


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Table 3. Model


Systems Studied Using the KIKO Mouse

System Reproductive Neuroendocrine Metabolic Skeletal Breast cancer Circadian

References 1, 30 –32 33–37 38 – 41 42– 49 32 50

uterine transcriptional regulation (9) (Figure 6A and Supplemental Figure 4). This indicates that ER␣ deficient in DNA-binding is not sufficient to mediate biological or transcriptional responses in uterine tissue as was reported for the liver of the EAAE mice (9), which lacked transcriptional responses as well. The implications of this finding in the larger context indicate a need to reexamine ER tethering in vivo. This mechanism was initially proposed to account for the effects of ER␣ on AP1-, SP-, or nuclear␬B-mediated responses in studies conducted in vitro with reporter plasmids that lack the complexity of chromatin that ER␣ and these other transcription factors normally encounter. Clearly, AP1 activity is affected by EAAE ER␣ in vitro (Figure 6B); however, in uterine tissues, ER␣-ERE binding might enable interactions between AP1 and ER␣ by altering chromatin structure. Thus, discovering whether our findings indicate that ER␣ tethering to nonERE motifs requires ER␣-ERE binding to permit the necessary ER␣ interactions with transcription factors or whether tethering occurs in vivo but lacks physiological significance will require further study. Ultimately, future ER␣ ChIP-seq analysis of EAAE tissues will indicate how loss of DNA-binding impacts ER␣ interaction with chromatin. Alternatively, the in vitro effects of ER␣ on AP1luc reporter activity could reflect an artifact such as sequestering of transcription factors or displacement of AP1. Some E2 responses, such as vascular dilation, are initiated from membrane-localized ER␣ (29), and because membrane localization results from palmitoylation of ER␣ C451, the EAAE ER␣ should retain these responses if they are independent from DNA-binding. When C451 was mutated to prevent palmitoylation, vasodilation was disrupted, whereas uterine proliferative and transcriptional responses were largely unaffected, indicating that ER membrane signaling is not the primary mechanism in operation in the uterus but greatly affects the vasculature (29). Thus, future studies will be performed to evaluate endpoints in EAAE mice to explore whether DNA-binding affects membrane ER␣-mediated responses.

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Our unexpected findings have demonstrated a defect in a model used by numerous laboratories in efforts to dissect effects mediated by direct or indirect interactions between ER␣ and target genes. We have established that the KIKO model introduces an aberrant mode of signaling to the tissues that has complicated interpretation of findings regarding mechanistic details of ER␣ signaling in reproductive, neuroendocrine, metabolic, skeletal, breast cancer and circadian studies (Table 3). Our previous studies and those of other investigators should be revisited to consider the novel activity we have uncovered here. Future work will require utilization of a truly HRE and ERE DNA-binding-deficient model, such as the EAAE mouse, to indicate the biological importance of tethered ER␣ signaling in physiological systems.

Acknowledgments We appreciate Dr Yukitomo Arao for his advice in cloning the EAAE mutation. We thank Dr Grace Kissling for statistical analysis of KIWT uterine weights, and Drs Heather Franco and Harriet Kinyamu for critical reading of our study. We thank members of the National Institute of Environmental Health Sciences (NIEHS) histology and microarray cores for their expertise. We appreciate the surgical expertise and husbandry provided by the NIEHS Comparative Medicine Branch. Address all correspondence and requests for reprints to: Sylvia Hewitt, 111 Alexander Drive, Research Triangle Park, NC 27709. E-mail: [email protected]. This work was supported in part by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health (Projects Z0170065 [to K.S.K.] and Z0101765 [to L.L.]) and National Institutes of Health (Grant 20 R01HD042311 [to F.J.D.]). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Disclosure Summary: The authors have nothing to disclose.

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are mediated by opposing actions of classical and nonclassical estrogen receptor pathways. J Bone Miner Res. 2005;20:1992–2001. Modder UI, Rudnik V, Liu G, Khosla S, Monroe DG. A DNA binding mutation in estrogen receptor-␣ leads to suppression of Wnt signaling via beta-catenin destabilization in osteoblasts. J Cell Biochem. 2012;113:2248 –2255. Syed FA, Fraser DG, Spelsberg TC, et al. Effects of loss of classical estrogen response element signaling on bone in male mice. Endocrinology. 2007;148:1902–1910. Syed FA, Fraser DG, Monroe DG, Khosla S. Distinct effects of loss of classical estrogen receptor signaling versus complete deletion of estrogen receptor alpha on bone. Bone. 2011;49:208 –216. Martin-Millan M, Almeida M, Ambrogini E, et al. The estrogen receptor-␣ in osteoclasts mediates the protective effects of estrogens on cancellous but not cortical bone. Mol Endocrinol. 2010;24:323– 334.

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Binding of the estrogen receptor DNA-binding domain to the estrogen response element induces DNA bending.

Uterine estrogen receptor interaction with estrogen-responsive DNA sequences in vitro: effects of ligand binding on receptor-DNA complexes.

DNA-binding.

The TEA domain: a novel, highly conserved DNA-binding motif.

Isolation of estrogen receptor-binding sites in human genomic DNA.

A novel DNA-binding motif abuts the zinc finger domain of insect nuclear hormone receptor FTZ-F1 and mouse embryonal long terminal repeat-binding protein.

The mouse glucocorticoid receptor DNA-binding domain is not phosphorylated in vivo.

Misperception of estrogen activity in patients treated with an estrogen receptor antagonist.

Generation of an estrogen receptor beta-iCre knock-in mouse.

Coregulator control of androgen receptor action by a novel nuclear receptor-binding motif.

In vivo monitoring of a cAMP-stimulated DNA-binding activity.

Effects of in vivo estrogen treatment on adipose tissue metabolism and nuclear estrogen receptor binding in isolated rat adipocytes.

Estrogen-related receptor γ is an in vivo receptor of bisphenol A.

The G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Nuclear Estrogen Receptor Activity and Stimulates Novel Phosphoproteomic Signatures.

Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor.

17β-estradiol regulates the expression of apolipoprotein M through estrogen receptor α-specific binding motif in its promoter.

Miz-1 activates gene expression via a novel consensus DNA binding motif.

A novel Arabidopsis DNA binding protein contains the conserved motif of HMG-box proteins.

The PilZ domain of MrkH represents a novel DNA binding motif.

The fork head domain: a novel DNA binding motif of eukaryotic transcription factors?

Differential DNA-binding abilities of estrogen receptor occupied with two classes of antiestrogens: studies using human estrogen receptor overexpressed in mammalian cells.

Opiate receptor: cooperativity of binding observed in brain slices.

In vitro binding of the estrogen receptor to DNA: absence of saturation at equilibrium.

Effect of estrogen receptor α binding on functional DNA methylation in breast cancer.