Sergio Onate

The steroid receptor coactivator-1 contains multiple receptor interacting and activation domains that cooperatively enhance the activation function 1 (AF1) and AF2 domains of steroid receptors.

Steroid receptors are ligand-inducible transcription factors, and their association with steroid receptor coactivators (SRCs) upon binding to DNA is necessary for them to achieve full transcriptional potential. To understand the mechanism of SRC-1 action, its ability to interact and enhance the transcriptional activity of steroid receptors was analyzed. First, we show that SRC-1 is a modular coactivator that possesses intrinsic transcriptional activity when tethered to DNA and that it harbors two distinct activation domains, AD1 and AD2, needed for the maximum coactivation function of steroid receptors. We also demonstrate that SRC-1 interacts with both the amino-terminal A/B or AF1-containing domain and the carboxyl-terminal D/E or AF2-containing domain of the steroid receptors. These interactions are carried out by multiple regions of SRC-1, and they are relevant for transactivation. In addition to the inherent histone acetyltransferase activity of SRC-1, the presence of multiple receptor-coactivator interaction sites in SRC-1 and its ability to interact with components of the basic transcriptional machinery appears to be, at least in part, the mechanism by which the individual activation functions of the steroid receptors act cooperatively to achieve full transcriptional activity.

The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences.

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.

Nuclear accessory factors enhance the binding of progesterone receptor to specific target DNA

The human progesterone receptor (PR) is dependent upon hormone and a nuclear accessory factor(s) for maximal binding to progesterone response elements (PRES) in vitro. Recombinant full-length PR, expressed in a baculovirus system and purified to apparent homogeneity, was used as a substrate to isolate and identify the accessory factor(s). The major PRE binding enhancement activity present in nuclear extracts was shown to be associated with the high mobility group chromatin protein HMG-1. Moreover, HMG-1 was equally effective in enhancing the DNA binding of both the A and B isoforms of PR. Enhancement of PRE binding was highly selective for HMG-1 as a single purified protein and was not mimicked by a general protein stabilization effect. In gel mobility shift assays, it appeared that HMG-1 enhanced PRE binding without stably participating as a component of the final DNA-PR complex, suggesting that HMG-1 acts indirectly by modifying the PR protein or the target DNA. HMG-1 is a sequence-independent DNA binding protein that recognizes distorted DNA structures and is also able to promote further distortions by bending DNA. Enhancement of PRE binding was found to be intrinsic to the conserved DNA binding domain of HMG-1 suggesting that HMG-1 acts by promoting a structural alteration in the target PRE-DNA.

Mechanisms Controlling Steroid Receptor Binding to Specific DNA Sequences

Mammalian progesterone receptors activated by hormone binding in nuclei of intact cells exhibit substantially higher binding activity for specific DNA sequences than receptors bound with hormone and activated in cell-free cytosol. Differences in DNA-binding activity occur despite the fact that both activated receptor forms sediment at 4S on sucrose gradients and are apparently dissociated from the heat shock protein 90. This suggests that hormone-induced release of heat shock protein 90 from receptors is necessary, but not sufficient for maximal activation of DNA binding. This report is a review of studies from our laboratories that have examined the role of receptor interaction with other nuclear protein factor(s), and receptor dimerization in solution, as additional regulatory steps involved in the process of receptor activation and binding to specific gene sequences.