Characterization of chromatin binding sites for different forms of uterine progesterone receptor

Abstract

We tested hamster uterine progesterone receptor (Rp) forms for binding to different chromatin preparations. Similar forms of chick oviduct Rp were used for comparison. Hamster Rp elutes from DEAE-Sephacel in the two peaks, peak I at 115 mM KC1 and peak II at 205 mM KC1. Chick Rp peaks I and II elute at 125 mM and 300 mM KC1, respectively. Both chick and hamster peak I displayed a higher level of binding to SDS-stripped chromatin (DNA) than to crude chromatin or 4 M guanidine hydrochloride (GuHCl)-extracted (nucleoacidic protein, NAP) chromatin while peak II bound 50% better to the NAP chromatin than to crude chromatin or DNA. 10 mM molybdate was used to stabilize Rp and to increase Rp recovery. Molybdate-stabilized hamster Rp elutes from DEAE at the peak II position and like peak II, binds poorly to DNA. Since molybdate prevents receptor activation, DNA-Rp interactions require activated Rp. Because molybdate did not prevent Rp binding to NAP chromatin, we conclude that both activated and unactivated Rp bind well to that matrix. Activated hamster Rp could be extracted from crude chromatin, NAP chromatin and DNA with 200 mM KC1. Unactivated Rp was extracted from NAP only with 6 M GuHCl or NaSCN, whereas KC1, glycerol or pyridoxal 5'-phosphate were not able to remove unactivated Rp from NAP. Various Rp forms did not compete with [ 3H]ORG 2058-Rp for binding to NAP but BSA did compete. Thus a large portion of Rp binding to NAP may represent nonspecific binding rather than binding to a finite number of Rp acceptor sites. These results suggest that the binding of activated Rp to crude chromatin may represent the actual acceptor sites in target cell nuclei. Since the high level of Rp binding sites in NAP chromatin may be an extraction artifact, the involvement of proposed masking proteins in regulating the availability of acceptor sites should be reconsidered. As an alternative to acceptor site regulation, changes in the Rp molecule itself may be important. Rp isolated from hamster uteri on days 1–4 of the estrous cycle was incubated with crude chromatin, NAP chromatin and DNA. The apparent level of Rp binding to chromatin and NAP chromatin increased 2.5-fold from day 1 to day 4, but Rp binding to DNA remained constant. This suggests that ovarian cycle-dependent changes occur in the unactivated Rp which affect its interactions with chromatin, and these changes disappear when receptor is activated. In summary, these studies indicate that the three matrices, crude chromatin-cellulose, NAP chromatincellulose, and DNA-cellulose, bind activated Rp in a KCl-extractable manner. Although NAP chromatin displayed increased salt-resistant Rp binding, such binding was not sensitive to competition with various forms of hormone-Rp complex and it was unaffected by Rp activation state. Our findings indicate that the previous concept of Rp acceptor sites in chromatin should be expanded to include another level of control which appears to involve the receptor protein itself.