Nuclease resistance and the enrichment of native nuclear acceptor sites for the avian oviduct progesterone receptor.

Abstract

High-affinity nucleoprotein acceptor sites for the avian oviduct progesterone receptor (PR) have been enriched by a combination of nuclease digestion and centrifugation. These enriched binding elements exhibited markedly enhanced PR binding on a per mass DNA basis compared to chromatin (20-to 25-fold) or dehistonized chromatin (4-to 5-fold). Electrophoretic analysis of the nuclease-resistant DNA showed that there is a set of DNA fragments of 100-150 base pairs that are protected from digestion. Excessive digestion resulted in smaller DNA fragments and a loss of PR binding activity. The PR binding was saturable using a crude receptor preparation and displayed a competition with the same receptor preparation that was labeled with nonradioactive progesterone. The enhanced binding was also demonstrable using highly purified receptor preparations that exhibit two classes of binding sites both of which are of high affinity and saturable as assessed by Scatchard analyses. These two high-affinity classes of binding sites are shown to be competed by unlabeled purified PR. The nuclease resistance of these nucleoprotein acceptor sites from chromatin is a property similar to the nuclear matrix binding sites suggesting a relationship between these two classes of nuclear acceptor sites.Based on the presently accepted mechanism of steroid hormone action, the interaction of a steroid receptor-hormone complex with the nucleus is an important step in the induction of specific gene products. A great deal of effort has been invested by a number of laboratories in trying to determine the relevant receptor-nuclear interactions. Despite these efforts, the exact nature of the nuclear binding sites (acceptor sites) is not completely understood. Steroid receptors have been shown to bind to DNA (1), RNA (2), chromatin (3), and ribonucleoprotein particles (4).This laboratory has been concentrating on the specific interaction ofthe chicken oviduct progesterone receptor (PR) with nuclear acceptor sites in oviduct chromatin. In contrast to the receptor binding to pure DNA sequences alone (5, 6), the binding of the oviduct PR to subfractions of chromatin containing nonhistone protein-DNA complexes, termed nucleoacidic protein or NAP, has been shown to be receptor dependent and saturable, of high affinity (7)(8)(9)(10)(11), to be receptor specific (12), and to generate patterns of bindings similar to that measured in vivo (13)(14)(15)(16). Further, the capacity of the PR to bind to nuclear acceptor sites reflects the ability of progesterone to alter RNA polymerase activity in nuclear run-off experiments (17, t) and to specifically induce the avidin gene (18). A specific subset of nonhistone proteins (acceptor proteins) has been shown to be necessary for the generation of specific nucleoprotein acceptor sites (10,11,(19)(20)(21).In other organisms, similar nuclear acceptor sites composed of tightly bound (to DNA) nonhistone proteins have been reported for the estrogen receptor/chicken oviduct system (17), for systems involving the estrog...

Interaction of two nonhistone proteins with the estradiol response element of the avian vitellogenin gene modulates the binding of estradiol-receptor complex.

“…A number of groups have presented evidence that specific NHPs together with the DNA were in some way involved in specifically binding the hormone-receptor complex to the chromatin and that naked DNA was unable to bind purified steroid receptors (24)(25)(26)(27)(28). Unfortunately, all these studies used only bulk chromatin and did not examine the role of specific DNA sequences in receptor binding.…”

Intranuclear mobility of estrogen receptor α and progesterone receptors in association with nuclear matrix dynamics

“…The nuclear mitotic apparatus protein (NuMA), first identified as a protein localizing on the spindle poles of mitotic cells, is a component of the nuclear matrix in interphase cells [Zeng et al, 1994;Harborth et al, 1999;Gribbon et al, 2002]. It has long been understood that steroid receptors associate with the nuclear matrix ligand-dependently [Hora et al, 1986;Barrack, 1987;Hu et al, 1994;Tang and DeFranco, 1996], and thus hypothesized that the nuclear matrix binding may play an important role in the regulation of steroid hormone receptor-mediated transcription. In the previous study, Stenoien et al [2000] visualized a nuclear matrix binding property of ERa using GFP-ERa, and showed that most of ligand-activated GFP-ERa was associated with the nuclear matrix by forming discrete clusters.…”

Immunohistochemical localization of the avian progesterone receptor and its candidate receptor binding factor (RBF‐1)

“…Selective removal of the chromatin protein fraction which contains RBF-1 results in the loss of the highest affinity class of PR binding sites. When RBF-1 is reconstituted to avian genomic DNA, the specific binding of PR is regenerated [Spelsberg et al, 1984Schuchard et al, 1991a,b;Hora et al, 1986;Goldberger et al, 1987;Rejman et al, 19911. The RBF-1 has been purified to homogeneity from oviduct nuclei and characterized.…”

“…Chromatin acceptor sites for the avian oviduct progesterone receptor (PR) have been studied extensively and found to consist of complexes of specific acceptor proteins tightly bound t o specific DNA sequences [Spelsberg et al, 1972[Spelsberg et al, , 1984[Spelsberg et al, , 1987a[Spelsberg et al, ,b, 1988Schuchard et al, 1991a,b;Pikler et al, 1976;Kon and Spelsberg, 1982;Hora et al, 1986;Goldberger et al, 1987;Goldberger and Spelsberg, 1988;Rejman et al, 19911. The specificity of this interaction is highlighted by in vivo and in vitro studies which demonstrate that the binding of PR to avian oviduct chromatin is not only saturable and high affinity [Spelsberg et al, 1983[Spelsberg et al, , 1984[Spelsberg et al, , 1987aPikler et al, 19761, but also receptor dependent [Pikler et al, 19761 and receptor specific [Spelsberg et al, 1987a,b;Kon and Spelsberg, 19821.…”