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.
Neo
Siew Jun
SJ
Institute of Materials Research and Engineering, Agency for Science, Technology and Research, 3 Research Link, Singapore 117602.
Su
Xiaodi
X
Thomsen
Jane S
JS