ESTROGEN-RECEPTOR ACCESSORY PROTEINS AUGMENT RECEPTOR-DNA INTERACTIONAND DNA BENDING

Increasing evidence suggests that accessory proteins play an important role in the ability of the estrogen receptor (ER) and other nuclear h ormone receptors to modulate transcription when bound to cis-acting ho rmone response elements in target genes. We have previously shown that four proteins, hsp70, protein disulfide isomerase (PDI) and two unkno wn proteins (p48 and p45), copurify with ER that has been isolated by site-specific DNA chromatography (BERE) and influence the interaction of ER with DNA in vitro. To better define the nature of these effects, we used filter binding and electrophoretic mobility shift assays to s tudy the ability of these proteins to alter the kinetics of ER-DNA int eraction and to influence the ability of ER to bend DNA when bound to an estrogen response element (ERE). The results of both assays indicat e that ERE-purified ER, with its four associated proteins (hsp70, PDI, p48, p45), has a greater ability to bind to the vitellogenin A2 ERE t han ER purified by estradiol-Sepharose chromatography in the absence ( ESeph) or presence (EATP) of ATP, in which p48, p45 (ESeph) and hsp70 (EATP) are removed. Surprisingly, the rates of association and dissoci ation of ER and ERE were essentially the same for all three mixtures, suggesting that one or more ER-associated proteins, especially p45 and p48, may be required for ER to attain maximum DNA binding activity. I n addition,circular permutation and phasing analyses demonstrated that the same ER-associated proteins produced higher order ER-DNA complexe s that significantly increased the magnitude of DNA distortion, but di d not alter the direction of the ER-induced bend of ERE-containing DNA fragments, which was toward the major groove of the DNA helix. These results suggest that p45 and/or p48 and possibly hsp70, play an import ant role both in the specific DNA binding and bending activities of ER and thus contribute to the overall stimulation of transcription in ta rget genes that contain cis-acting EREs. (C) 1997 Elsevier Science Ltd . All rights reserved.