HIGH-MOBILITY GROUP (HMG) PROTEIN HMG-1 AND TATA-BINDING PROTEIN-ASSOCIATED FACTOR TAF(II)30 AFFECT ESTROGEN RECEPTOR-MEDIATED TRANSCRIPTIONAL ACTIVATION

The estrogen receptor (ER) belongs to a family of ligand-inducible nuc lear receptors that exert their effects by binding to cis-acting DNA e lements in the regulatory region of target genes. The detailed mechani sms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER- ERE interaction and transcription initiation, we employed purified rec ombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacte rial systems. The effect of high-mobility group (HMG) protein HMG-1 an d purified recombinant TATA-binding protein-associated factor TAF(II)3 0 on ER-ERE binding and transcription initiation were assessed by elec trophoretic mobility shift assay and in vitro transcription from an ER E-containing template (pERE(2)LovTATA), respectively. We find that pur ified, recombinant ER fails to bind to ERE in spite of high ligand-bin ding activity and electrophoretic and immunological properties identic al to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and pro motes ER-ERE binding in a concentration- and time-dependent manner. Th e effectiveness of HMG-1 to stimulate ER-ERE binding in the electropho retic mobility shift assay depends on the sequence flanking the ERE co nsensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE bind ing, it fails to stimulate transcription initiation either in the pres ence or absence of hormone. In contrast, TAF(II)30, while not affectin g ER-ERE binding, stimulates transcription initiation 20-fold in the p resence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by t he latter to stimulate transcription initiation.