Identification and characterization of estrogen receptor-regulated gene expression programs

The physiological effects of natural and synthetic estrogens are mediated by estrogen receptor alpha (ER alpha), and estrogen receptor beta (ER beta). Within the nucleus of target cells, ER alpha and ER beta serve as ligand-activated transcription factors to stimulate or repress the transcription of estrogen receptor regulated genes. ER alpha and ER beta may be co-expressed in estrogen-responsive cells, but may also be differentially expressed in a cell- and tissue-specific manner. In addition, within a given context these two receptors have different ligand binding and transcriptional activities. Taken together, these attributes underlie differences in target gene regulation, and overall, different physiological actions by ER subtypes. The work described here is an attempt to understand the roles of ER alpha and ER beta in target tissues (e.g. bone, breast, uterus) including the gene networks and cell signaling pathways under ER regulation. We have also characterized the regulation of one of the ER-regulated genes, Carbonic Anhydrase XII, and examined its regulation by ER alpha through use of a conserved distal enhancer. The work described here reports the characterization of individual gene regulatory actions of ER alpha and ER beta. To investigate the individual actions of ER alpha or ER beta, we utilized Affymetrix oligonucleotide arrays to profile transcripts regulated by 17beta-estradiol (E2) in U2OS-ER alpha and U2OS-ER beta cells. These cell lines were constructed by stable integration of ER alpha or ER beta into human osteoblast-like U2OS osteosarcoma cells and initially characterized for ER subtype expression, E2-binding, and cellular responses to E2, including proliferation, motility, and adhesion. Cells expressing apo-ER alpha or apo-ER beta did not show significant alteration in adhesion or proliferation after addition of E2, however there was a significant stimulation of migration in E2-treated ER beta-expressing cells. U2OS-ER alpha, and U2OS-ER beta cells were treated with 10 nM E2 for 0, 4, 8, 24, and 48 hours and total RNA was collected and hybridized to Affymetryx U95Av2 GeneChips and subjected to a Confidence Score to determine E2-regulated RNAs. Of the ca. 100 stimulated or repressed genes identified, some were stimulated by E2 equally through ER alpha and ER beta, whereas others were selectively stimulated via ER alpha or ER beta. The E2-regulated genes showed three distinct temporal patterns of expression over the 48 hour time course studied. Among stimulated genes, ER alpha-containing cells exhibited a greater number of regulated transcripts, and overall magnitude of stimulation was increased as compared those regulated by ER beta. Of the functional categories of the E2-regulated genes, most numerous were those encoding cytokines and factors associated with immune response, signal transduction, and cell migration and cytoskeleton regulation, indicating that E2 can exert effects on multiple pathways in these osteoblast-like cell lines. Of note, E2 up-regulated several genes associated with cell motility selectively via ER beta, in keeping with the selective E2 enhancement of the motility of ER beta-containing cells. On genes regulated equally by E2 via ER alpha or ER beta, the phytoestrogen genistein preferentially stimulated gene expression via ER beta. These studies indicate both common as well as distinct target genes for these two ERs, and identify many novel genes not previously known to be under estrogen regulation. We have examined the ER regulation of the Carbonic Anhydrase XII (CA12) gene, a gene identified as E2-regulated in the studies described above. We investigated the expression of CA12 and its and regulation of by 17beta-estradiol and selective estrogen receptor modulators in breast cancer cells, and characterize the ER usage of a distal enhancer necessary for CA12 gene regulation. We find that CA12 expression is highly correlated with ER alpha expression in human breast tumors. We demonstrate that E2 and SERMS increase CA12 mRNA and protein in multiple breast cancer cell types expressing ER alpha, and that CA12 regulation by estrogen is a primary transcriptional response mediated by ER alpha. By genome-wide chromatin immunoprecipitation (ChIP) and ChIP scanning of the CA12 locus, we find E2-occupied ER alpha is recruited to a distal region 6.1 kb upstream of the CA12 transcription start site (TSS) in vivo. We find that E2 treatment results in recruitment of RNA polymerase II and steroid receptor coactivators SRC-2 and SRC-3 to the CA12 genomic locus and is correlated with increased histone H4 acetylation. Mutagenesis of an imperfect estrogen-responsive element within this -6.1kb distal enhancer region abolishes estrogen-dependent heterologous reporter activity. Chromosome conformation capture (3C) and chromatin immunoprecipitation assays demonstrate that this distal enhancer communicates with the transcriptional start site of the CA12 gene via intra-chromosomal looping upon hormone treatment. This distal enhancer element is observed in the homologous mouse genomic sequence, and the expression of the mouse homolog, Car12, is rapidly and robustly stimulated by estradiol in the mouse uterus in vivo, suggesting that the ER regulation of CA12 is mechanistically and evolutionarily conserved. Our findings highlight the crucial role of ER in regulation of the CA12 gene, and provide insight into the transcriptional regulatory mechanism that accounts for the strong association of CA12 and ER in human breast cancers. In addition, our findings imply that involvement of long distance enhancers in regulation of estrogen-responsive genes in breast cancer may be more frequent than previously appreciated