Isolation and characterization of the nuclear acceptor that binds the progesterone‐receptor complex in hen oviduct

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Abstract
Progesterone binds to the nuclear chromatin of hen oviduct cells within minutes after its administration. This is followed by alterations in the RNA polymerase activities (15–30 min after treatment) and the appearance of specific messenger RNA (2 hr after treatment). It appears that the first nuclear event resulting in changes in gene expression represents the chromatin binding. Further characterization of this nuclear interaction, involving the administration of multiple doses of progesterone to estrogen‐treated immature chicks, revealed the presence of several classes of nuclear binding sites in the oviduct. Comparisons of serum progesterone levels and responses of RNA polymerase I and II activities with the nuclear binding indicated that the highest‐affinity classes of sites (∼ 10,000 sites per cell) are the biologically important ones. Binding studies in vitro, using isolated progesterone‐receptor complex, also reveal the presence of these multiple classes of sites. The highest‐affinity class of nuclear sites measured in vitro represented 6,000–10,000 sites per cell with Kd∼10‐9 M. Although these sites appeared to be tissue‐specific, similar high‐affinity sites appeared to be present but were completely masked in the chromatin of nontarget organs (spleen and erythrocyte). Even the target tissue (oviduct) chromatin appeared to have 70% masking. Further fractionation studies involving DNA affinity chromatography with chromatin bound to cellulose or hydroxylapatite revealed that these partially purified acceptor proteins were bound to DNA with very high affinity. The “acceptor proteins” must be reannealed to DNA to achieve binding activity. Preliminary evidence is presented which suggests that the acceptors reanneal to specific sequences on the DNA. These results support the idea that acidic proteins determine the high‐affinity nuclear binding sites for steroids.