Surface plasmon resonance study of cooperative interactions of estrogen receptor alpha and transcriptional factor Sp1 with composite DNA elements.

We have applied surface plasmon resonance (SPR) spectroscopy to study the cooperative interactions of estrogen receptor alpha (ERalpha) and transcription factor Sp1 with a composite DNA element, containing an estrogen response element (ERE) half-site upstream of two adjacent Sp1 sites (+571 ERE/Sp1 composite site in promoter A of the human PR gene). Using nuclear extracts of MCF-7 breast cancer cells as sample, we have shown that Sp1 is associated with Sp1-binding sites only, whereas ERalpha can be recruited to DNA both through direct binding to the ERE half-site and/or through protein-protein interactions with DNA-bound Sp1. The ERE half-site and the proximal Sp1 site are only 4 bp apart, and our data suggests that one transcription factor bound to DNA constitutes a sterical hindrance of the accessibility of the binding site for the other transcription factor. Our data confirms previous observations that ERalpha increases the amount of Sp1 recruited to the composite binding site in a dose-dependent manner. Using recombinant proteins, we have unambiguously proved the formation of a ternary complex of ERalpha/Sp1-composite DNA, for which previously published electrophoretic mobility shift assay (EMSA) results are contradictive. With this study, we have demonstrated that the solid-liquid-phase SPR assay is a powerful alternative for studying multiprotein-DNA interactions and is superior to the EMSA experiments as it is capable of real-time measurements, can quantify the amount of protein bound, and can capture transient and weak binding interactions. The comprehensive characterization of the synergistic interactions between ERalpha-DNA, Sp1-DNA, and ERalpha-Sp1 contributes to the understanding of how ERalpha and Sp1 influence and activate gene transcription.

A two-step antibody strategy for surface plasmon resonance spectroscopy detection of protein-DNA interactions in nuclear extracts.

Surface plasmon resonance (SPR) spectroscopy has emerged as a powerful alternative to conventional biochemistry methods for studying protein-DNA interactions that involve recombinant proteins of known identity. There are, however, limited demonstrations of SPR detection of protein-DNA bindings in crude samples, e.g., cell extracts, where the challenge is to detect and identify specific DNA binding protein(s) among other protein components in a physiological setting. We have developed a two-step antibody approach for an SPR study of estrogen receptor alpha (ERalpha)-DNA interactions, in which nuclear extracts prepared from MCF-7 breast cancer cells were used as the source of ERalpha protein. Following the binding of nuclear extracts to surface-immobilized estrogen response elements, rabbit anti-ERalpha antibody followed by a secondary antibody (goat anti-rabbit IgG) were applied to recognize the bound ERalpha and amplify the signals, respectively. Through a series of experiments, we have demonstrated that the magnitude of the binding signals from the secondary antibody reflects the affinity by which ERalpha binds to different DNA sequences. The detection sensitivity is determined by the amount of nuclear extracts and the concentration of primary antibody used. The sequence specificity of the nuclear ERalpha measured using the two-step antibody approach is in agreement with that measured for recombinant ERalpha protein (using receptor binding signals).