Reconstitution of nativelike nuclear acceptor sites of the avian oviduct progesterone receptor: evidence for involvement of specific chromatin proteins and specific DNA sequences

Abstract

A specific fraction of avian oviduct chromosomal proteins can be reannealed to pure avian DNA to reconstitute nativelike specific nuclear binding sites (acceptor sites) for the oviduct progesterone receptor (PR). These specific nuclear binding sites represent the difference between the binding to the reconstituted NAP and that to pure DNA. The specific fraction of chromatin protein which contains the acceptor activity, fraction CP-3, is very tightly bound to hen DNA in a complex termed nucleoacidic protein (NAP). Removal of the CP-3 fraction from NAP results in a loss of specific PR binding sites. Resins containing chromatin adsorbed to hydroxylapatite are used as a rapid method to isolate the CP-3 fraction. Reconstitution of the CP-3 fraction to DNA by the described method involving a regressing gradient of 6-0 M guanidine hydrochloride (Gdn-HCl) results in a reconstituted NAP which displays specific PR binding sites identical with those in native (undissociated) NAP and whole chromatin. Optimal conditions and potential problems for reconstituting these nucleoproteins are described. Only partially purified receptor preparations were used in these cell-free binding analyses since they have been shown to bind with similar properties and patterns as the nuclear binding in vivo. Therefore, the binding of PR to the reconstituted NAPs was demonstrated to be receptor dependent, saturable, and of high affinity. Further, the pattern of binding to the reconstituted sites mimics those which are observed in vivo. Thus, nonfunctional receptors that cannot translocate and bind to the nuclear acceptor sites in vivo also failed to bind to the acceptor sites on the reconstituted NAPs generated by the acceptor proteins. In contrast, the binding to pure DNA does not reflect these receptor differences in receptor bindings. Specific binding of PR to reconstituted NAP can be reversed by again removing the protein fraction. Moreover, the specific binding can be destroyed by proteases and protected by protease inhibitors, indicating that acceptor activity is proteinaceous in nature. The reconstitution of the activity is both a concentration-dependent and time-dependent process. During the reconstitution, acceptor activity appears to reconstitute on the DNA when the Gdn-HCl concentration reaches 2.0 M. By use of the reconstitution method as an assay for acceptor activity, the activity in the CP-3 fraction was shown by molecular sieve chromatography to elute in a relatively broad molecular weight range between 13 000 and 25 000. The activity also focuses in isoelectric focusing resins with apparent pI's of 5.2 and 6.4.(ABSTRACT TRUNCATED AT 400 WORDS)

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

“…Similar binding results were observed using the milder (DNase/exonuclease/Sl nuclease) digestion method. Thus, the saturable, high-affinity binding of PR to NAPf displayed similar properties as did the PR binding to intact NAP (8,9,11) or chromatin (7,36).…”

“…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).…”

“…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).…”

“…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 estrogen and progesterone receptors in sheep brain (22), in hamster uteri (t), in cow, rabbit, and human uteri (21, 23, 24), for systems involving the glucocorticoid receptor in both rat liver (25) and human leukemic cell line system (26), and finally for the androgen receptor rat prostate system (27). Thus, the acceptor protein-DNA complex as a potential nuclear acceptor site model for steroid receptors seems to be universal.…”

Evidence for specific DNA sequences in the nuclear acceptor sites of the avian oviduct progesterone receptor.

“…Similarly, a role for tightly bound proteins and DNA in the nuclear acceptor sites for androgens, estrogens, progesterones, and glucocorticoids in mammalian tissues has been reported (12,(14)(15)(16)(17). It has recently been shown that the number of PR binding sites on hen DNA, generated by rehybridizing increasing quantities of acceptor protein to the hen DNA, is limited (6), suggesting that specific DNA sequences may be involved in the nuclear acceptor sites for PR. In this paper, we attempt to substantiate this possibility by determining whether or not DNA from other species can bind to the hen oviduct acceptor protein to generate nuclear acceptor sites for the PR.…”