Design of estrogen response element (ERE) binding proteins to study the roles of ERE-containing genes in cell proliferation and the functional differences between ER alpha and ER beta.

摘要

Estrogen receptor alpha (ERalpha) and beta (ERbeta) mediate estrogen signaling through genomic and non-genomic pathways. Although the interaction of ERs with estrogen response elements (EREs) constitutes a critical genomic signaling for the regulation of estrogen responsive genes, the relative importance of ERE-containing genes in the proliferation of breast cancer cells is unclear. To address this issue, we constructed a series of ERE binding activators (EBAs) and repressors (EBRs) by a genetic conjugation approach. Biochemical and transfection studies revealed that EBAs activate and EBRs repress the transcription of ERE-containing genes. Flow cytometry analysis showed that EBRs block the proliferation of ER-positive MCF-7 cells. Interestingly, although EBAs induce the GUS phase transition in MCF-7 cells, they inhibit the transition in ER-negative MDA-MB-231 cells. Moreover, the effects of EBAs and EBRs on the G1/S phase transition were abolished by the introduction of mutations that disrupt their DNA binding abilities. Taken together, our studies support the view that ERE-containing genes play major roles in the proliferation of breast cancer cells.; It is established that ERbeta displays less potency than ERalpha to induce transcription in the ERE-dependent genomic signaling. However, mechanisms underlying the functional differences between ERs are unclear. Previous studies showed that the removal of the N-terminus of ERbeta augments the ability of ERbeta to enhance transcription. One possibility is that the N-terminus of ERbeta inhibits the ERbeta-ERE interaction. To investigate the role of N-terminus of ERbeta in ERE binding in situ, we utilized an EBA and developed an in situ competition assay. Using this assay, we found that the N-terminus of ERbeta, not of ERalpha, significantly reduces the ERE binding. Interestingly, the N-terminus of ERbeta does not alter the intrinsic DNA binding affinity but sequesters a large population of the receptor in an inactive pool that is incompetent to bind to ERE. Moreover, we found that the transactivation ability of the N-terminal truncated ERbeta strongly correlates with its ERE binding activity. Thus, it appears that the N-terminus of ERbeta impairs the ability of the receptor to interact with an ERE and consequently contributes to the weak transactivation capacity of the receptor.