Location analysis for the estrogen receptor-alpha reveals binding to diverse ERE sequences and widespread binding within repetitive DNA elements

Abstract

Location analysis for estrogen receptor-alpha (ERalpha)-bound cis-regulatory elements was determined in MCF7 cells using chromatin immunoprecipitation (ChIP)-on-chip. Here, we present the estrogen response element (ERE) sequences that were identified at ERalpha-bound loci and quantify the incidence of ERE sequences under two stringencies of detection: <10% and 10-20% nucleotide deviation from the canonical ERE sequence. We demonstrate that approximately 50% of all ERalpha-bound loci do not have a discernable ERE and show that most ERalpha-bound EREs are not perfect consensus EREs. Approximately one-third of all ERalpha-bound ERE sequences reside within repetitive DNA sequences, most commonly of the AluS family. In addition, the 3-bp spacer between the inverted ERE half-sites, rather than being random nucleotides, is C(A/T)G-enriched at bona fide receptor targets. Diverse ERalpha-bound loci were validated using electrophoretic mobility shift assay and ChIP-polymerase chain reaction (PCR). The functional significance of receptor-bound loci was demonstrated using luciferase reporter assays which proved that repetitive element ERE sequences contribute to enhancer function. ChIP-PCR demonstrated estrogen-dependent recruitment of the coactivator SRC3 to these loci in vivo. Our data demonstrate that ERalpha binds to widely variant EREs with less sequence specificity than had previously been suspected and that binding at repetitive and nonrepetitive genomic targets is favored by specific trinucleotide spacers.

Figure 1.

ChIP-PCR validation of ERα-bound loci. ERα-bound loci determined by ChIP-on-chip were interrogated for E2-dependent ERα binding using quantitative ChIP-PCR (for genomic coordinates see Table 2). MCF7 cells were treated with E2 (100 nM) or vehicle (EtOH) for 45 min. ChIP was performed using antibodies against ERα. Quantitative PCR using genomic primers employed the 28S rRNA coding sequence as internal reference. Shown are E2-treated values normalized to control values. ERα was not recruited to an ERE-less and ChIP-on-chip negative locus dubbed ERE(–). Similar studies using genomic PCR primers for the known E2-responsive genes TFF1 and CTSD demonstrated rapid recruitment of ERα to these enhancer regions in response to E2. ChIP-PCR targeting 17 additional loci similarly revealed E2-dependent enrichment (>2-fold) of ERα at all sites. Values are the average of three experiments with SEM. Oligonucleotide sequences are presented in Supplementary Table S1.

Figure 2.

The canonical 280-bp Alu sequence is composed of two monomers, derived from the 7SL RNA gene, separated by an adenosine rich connector. The consensus AluSc sequence (A) contains an ERE-like sequence (red) identified in the 3′ monomer of the element. A similar element (blue) also occurs in the 5′ monomer of the Alu element. The monomers are separated by an A-rich connector sequence (underlined). The previously indentified Alu-Retinoic Acid Receptor response element (RARE) sequence is also indicated (purple). (B) Diverse ERE-like sequences located in the 3′-monomers of classical Alu element family sequences are indicated. Mismatches from the consensus ERE sequence are indicated by lower case letters.

Figure 3.

Electrophoretic mobility shift assay (EMSA) of ERα binding to nonconsensus repetitive element ERE sequences. EMSA was performed using HESC cell nuclear lysates plus recombinant ERα and labeled consensus ERE probe (lanes 1–3) or repetitive DNA element ERE sequences as probes: B68 (lanes 4–6) and D66 (lanes 7–9). An ERα-containing complex bound to all three ERE sequences (arrowhead, lanes 1, 4 and 7) and was confirmed by supershift (bracket) using a monoclonal antibody that recognizes ERα (lane 2). Specific ERα-containing complexes were similarly shown by loss of bands for B68 (lane 5) and D66 (lane 8) when anti-ERα antibody was added. The nonspecific antibody recognizing Sp1 had no effect on the ERα-containing complexes bound to the classical ERE (lane 3) and B68 (lane 6), but displayed moderate effects on complexes bound to D66 (lane 9), indicating that Sp1 may cooperate with ERα in binding to this sequence. Mismatches from the consensus ERE sequence are indicated by lower case letters and red font. Oligonucleotide sequences are presented in Supplementary Table S1.

Figure 4.

Luciferase reporter assays of cloned genomic loci bound by ERα and displaying high enhancer activity in MCF7 cells. Genomic loci (average length 776 bp) containing one or more ERE sequences were cloned into a minimal promoter reporter construct driving luciferase gene expression. Specific wild-type ERE sequences and mutated (i.e. E1M) ERE sequences are indicated above each clone being tested (repetitive element EREs are indicated by red font, mismatches from the classical ERE sequence are indicated by lower case lettering, and mutated sequences were changed to tttttt where indicated). Basal and E2-stimulated luciferase values are shown normalized to co-transfected β-galactosidase-expressing plasmid. Clone D54 contains three ERE sequences and the third is in a repetitive element (see Table 2) which contributes to overall enhancer function (A). B68 contains two ERE sequences both of which reside within repetitive DNA elements and both contribute to enhancer function (B). D70 also contains two ERE sequences both of which reside within repetitive DNA elements (C). Values are the average of three experiments, performed in triplicate, with SEM indicated. *P < 0.01 comparing E2-treated wild type with E2-treated mutated reporter. ^§P < 0.01 comparing E2-treated empty vector with E2-treated mutated reporter. ^†P < 0.01 comparing vehicle control-treated with E2-treated reporter.

Figure 5.

Luciferase reporter assays of cloned genomic loci bound by ERα and displaying moderate, low, and no enhancer activity in MCF7 cells. Genomic loci (average length 776 bp) containing one or more ERE sequences were cloned into a minimal promoter reporter construct driving luciferase gene expression. Specific wild-type ERE sequences and mutated (i.e. E1M) ERE sequences are indicated above each clone being tested (repetitive element EREs are indicated by red font, mismatches from the classical ERE sequence are indicated by lower case lettering, and mutated sequences were changed to tttttt where indicated). Basal and E2-stimulated luciferase values are shown normalized to co-transfected β-galactosidase-expressing plasmid. D75 contains two repetitive element ERE sequences responsible for enhancer function (A). C31 contains two, nonrepetitive element EREs that are functional (B). Additional ERE-containing clones with low or no enhancer function in luciferase assays are indicated (C). Values are the average of three experiments, performed in triplicate, with SEM indicated. *P < 0.01 comparing E2-treated wild-type with E2-treated mutated reporter. ^§P < 0.01 comparing E2-treated empty vector with E2-treated mutated reporter. ^†P < 0.01 comparing vehicle control-treated with E2-treated reporter.

Figure 6.

ChIP-PCR confirms estrogen-dependent recruitment of SRC3 to repetitive element ERE-containing genomic loci. MCF7 cells were treated with E2 (100 nM) or vehicle (EtOH) for 45 min. ChIP was performed using antibodies against SRC3. Quantitative PCR using genomic primers employed the 28S rRNA coding sequence as internal reference. Shown are E2-treated values normalized to control values. SRC3 was not recruited to an ERE-less and ERα ChIP-negative locus dubbed ERE(–). Similar studies using genomic PCR primers for the known E2-responsive gene TFF1 demonstrated rapid recruitment of SRC3 to this enhancer region in response to E2. ChIP-PCR targeting seven additional loci revealed E2-dependent enrichment (>2-fold) of SRC3 at all sites. B68, D54, D66, D70, D75 and K31 are all genomic loci that demonstrate repetitive element ERE sequences. Values are the average of four experiments with SEM. Oligonucleotide sequences are presented in Supplementary Table S1.