Differential regulation of native estrogen receptor-regulatory elements by estradiol, tamoxifen, and raloxifene

Abstract

Estrogen receptors (ERs) regulate gene transcription by interacting with regulatory elements. Most information regarding how ER activates genes has come from studies using a small set of target genes or simple consensus sequences such as estrogen response element, activator protein 1, and Sp1 elements. However, these elements cannot explain the differences in gene regulation patterns and clinical effects observed with estradiol (E(2)) and selective estrogen receptor modulators. To obtain a greater understanding of how E(2) and selective estrogen receptor modulators differentially regulate genes, it is necessary to investigate their action on a more comprehensive set of native regulatory elements derived from ER target genes. Here we used chromatin immunoprecipitation-cloning and sequencing to isolate 173 regulatory elements associated with ERalpha. Most elements were found in the introns (38%) and regions greater than 10 kb upstream of the transcription initiation site (38%); 24% of the elements were found in the proximal promoter region (<10 kb). Only 11% of the elements contained a classical estrogen response element; 23% of the elements did not have any known response elements, including one derived from the naked cuticle homolog gene, which was associated with the recruitment of p160 coactivators. Transfection studies found that 80% of the 173 elements were regulated by E(2), raloxifene, or tamoxifen with ERalpha or ERbeta. Tamoxifen was more effective than raloxifene at activating the elements with ERalpha, whereas raloxifene was superior with ERbeta. Our findings demonstrate that E(2), tamoxifen, and raloxifene differentially regulate native ER-regulatory elements isolated by chromatin immunoprecipitation with ERalpha and ERbeta.

Figure 1

Distances of Fragments from the Nearest Gene in the ChIP-CS Library Are Widely Distributed Distances of each fragment to the nearest TSS (Tables 1 and 2) were plotted on a histogram. The y-axis represents the number of the clones, whereas the x-axis represents the distance of the clone to the nearest TSS.

Figure 2

ERα Interacts with Elements from the ChIP-CS Library Tetracycline-inducible U2OS-ERα cells were treated with 10 nm E₂ for 3 h, and ChIP was performed using antibodies to ERα. Immunoprecipitated chromatin fragments were PCR amplified and quantitated by real-time PCR using primers for the CECR6, SPATA13, NKD, LLGL2, H19, and NKG2 elements. Cont, Control.

Figure 3

E₂ Recruits p160 Coactivators to Genes from the ChIP-CS Library Tetracycline-inducible U2OS-ERα cells were treated with 10 nm E₂ for increasing times, and ChIP was performed using antibodies to the p160 coactivators, SRC-1, SRC-2, or SRC-3. Immunoprecipitated chromatin fragments were PCR amplified and quantitated by real-time PCR using primers for the CECR6, SPATA13, NKD, LLGL2, H19, and NKG2 elements (panels A–F, respectively).

Figure 4

ERβ Activates the CECR6 and NKD Genes and Recruits p160 Coactivators Tetracycline-inducible U2OS-ERβ cells were treated with 10 nm E₂ for increasing times. CECR6 (A) or NKD (B) mRNA was measured by qPCR. Each data point is the average of triplicate determinations ± sem. The asterisk represents a significant difference from control (P < 0.05). ChIP assays were performed using antibodies to ERβ (C and D) and the p160 coactivators, SRC-1, SRC-2, or SRC-3 (E and F). Immunoprecipitated chromatin fragments were PCR amplified and quantitated by real-time PCR using primers for the CECR6 (C and E) and NKD (D and F) elements. Cont, Control.