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

• Published in: Biochemistry (Easton) 1984-10, Vol.23 (22), p.5103-5113
• Main Authors: Spelsberg, Thomas C, Gosse, Barbara J, Littlefield, Bruce A, Toyoda, Hiroo, Seelke, Ralph
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Biological and medical sciences; Cell receptors; Cell structures and functions; Chickens; Chromatin - metabolism; Chromatography, Gel; DNA - metabolism; Female; Fundamental and applied biological sciences. Psychology; Isoelectric Focusing; Kinetics; Molecular and cellular biology; Nucleoproteins - metabolism; Oviducts - analysis; Progesterone - metabolism; Receptors, Progesterone - metabolism; Seasons
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00317a004

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