Nuclear association states of rat uterine oestrogen receptors as probed by nuclease digestion.

Abstract

The solubilization of oestrogen receptors from uterine nuclei by micrococcal nuclease and deoxyribonuclease I was examined after the injection of oestradiol or Nafoxidine into castrated female rats. At 1h after an injection of oestradiol, 30% (0.18pmol/mg of DNA) of the nuclear oestrogen receptors was solubilized by 5 min of mild digestion with either nuclease. No further receptor release occurred, although DNA hydrolysis continued throughout a 20min interval. The limitation in receptor solubilization was not due to an artifact of digestion conditions or insufficient nuclease concentrations. Similar patterns of receptor solubilization and DNA hydrolysis were obtained with both nucleases whether the animals had been injected with oestradiol 1h before death or if the uteri from uninjected animals were incubated with [(3)H]oestradiol for 1h in vitro. When uterine nuclei were digested with these enzymes 12h after the animal was injected with oestradiol there was little change in the quantity of nuclease-sensitive sites (0.11pmol/mg of DNA); however, the quantity of nuclease-resistant sites decreased 10-fold. These values correspond quantitatively to the changes in salt-resistant and salt-extractable sites observed over a 12h interval after oestradiol treatment. Nuclease digestion of uterine nuclei obtained 16h after Nafoxidine treatment gave a pattern qualitatively and quantitatively similar to that observed 1h after oestradiol treatment, a result consistent with the agonist/antagonist action of this compound. An analysis by sucrose-density-gradient centrifugation of the time course of nuclease-dependent receptor solubilization indicated that the solubilized receptors were not associated with discrete nucleosomal fragments. We believe that these data indicate that only a portion of the receptors translocated to the nucleus become associated with chromatin, and this association may occur on regions of chromatin that are preferentially susceptible to nucleolytic cleavage.