title: The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences.
abstract: Steroid hormone receptors are ligand-dependent transcriptional activators that exert their effects by binding as dimers to cis-acting DNA sequences termed hormone response elements. When human progesterone receptor (PR), expressed as a full-length protein in a baculovirus system, was purified to homogeneity, it retained its ability to bind hormonal ligand and to dimerize but exhibited a dramatic loss in DNA binding activity for specific progesterone response elements (PREs). Addition of nuclear extracts from several cellular sources restored DNA binding activity, suggesting that PR requires a ubiquitous accessory protein for efficient interaction with specific DNA sequences. Here we have demonstrated that the high-mobility-group chromatin protein HMG-1, as a highly purified protein, dramatically enhanced binding of purified PR to PREs in gel mobility shift assays. This effect appeared to be highly selective for HMG-1, since a number of other nonspecific proteins failed to enhance PRE binding. Moreover, HMG-1 was effective when added in stoichiometric amounts with receptor, and it was capable of enhancing the DNA binding of both the A and B amino-terminal variants of PR. The presence of HMG-1 measurably increased the binding affinity of purified PR by 10-fold when a synthetic palindromic PRE was the target DNA. The increase in binding affinity for a partial palindromic PRE present in natural target genes was greater than 10-fold. Coimmunoprecipitation assays using anti-PR or anti-HMG-1 antibodies demonstrated that both PR and HMG-1 are present in the enhanced complex with PRE. HMG-1 protein has two conserved DNA binding domains (A and B), which recognize DNA structure rather than specific sequences. The A- or B-box domain expressed and purified from Escherichia coli independently stimulated the binding of PR to PRE, and the B box was able to functionally substitute for HMG-1 in enhancing PR binding. DNA ligase-mediated ring closure assays demonstrated that both the A and B binding domains mediate DNA flexure. It was also demonstrated in competition binding studies that the intact HMG-1 protein binds to tightly curved covalently closed or relaxed DNA sequences in preference to the same sequence in linear form. The finding that enhanced PRE binding was intrinsic to the HMG-1 box, combined with the demonstration that HMG-1 or its DNA binding boxes can flex DNA, suggests that HMG-1 facilitates the binding of PR by inducing a structural change in the target DNA.
authors.affiliation: Department of Pathology, University of Colorado Cancer Center, Denver 80262.
authors.forename: S A
authors.initials: SA
authors.lastname: Oñate
authors.forename: P
authors.initials: P
authors.lastname: Prendergast
authors.forename: J P
authors.initials: JP
authors.lastname: Wagner
authors.forename: M
authors.initials: M
authors.lastname: Nissen
authors.forename: R
authors.initials: R
authors.lastname: Reeves
authors.forename: D E
authors.initials: DE
authors.lastname: Pettijohn
authors.forename: D P
authors.initials: DP
authors.lastname: Edwards
mesh_terms: D000818:Animals; D016367:Baculoviridae; D001665:Binding Sites; D001667:Binding, Competitive; D001943:Breast Neoplasms; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006609:High Mobility Group Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D009036:Moths; D011980:Receptors, Progesterone / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D046911:Macromolecular Substances; D011980:Receptors, Progesterone; D004247:DNA