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An analysis of the binding of the chick oviduct progesterone-receptor to chromatin
Abstract:
The binding of progesterone-receptor complexes to chromatin from target and nontarget tissues was studied in vitro. Chromatin from both target and nontarget tissues responds in a similar manner to saly and cofactors and has the same K(D) (approx. 3.10(-9) M) for the progesterone-receptor complex. The only observed difference in the binding of the progesterone-receptor complex to target and nontarget chromatins is the difference in total number of acceptor sites. oviduct chromatin has approx. 1300 sites/pg DNA, spleen chromatin has approx. 840 sites/pg DNA, and erythrocyte chromatin has about 330 sites/pg DNA. The K(D) and number of acceptor sites for progesterone-receptor complex binding to oviduct chromatin remains the same even after extensive purification of the progesterone-receptor complex. Activation of cytosol labeled with [3H]progesterone by preincubation at 25 degrees C, analogous to that required for maximal nuclear binding, occurs if the binding studies to chromatin are performed in 0.025 M salt. The absence of an observable temperature effect when the studies are performed at 0.15 M salt is due to the activation of the receptor by salt. The dissociation of the progesterone-receptor complex from chromatin exhibits a single dissociation rate and the initial event is the appearance of free progesterone rather than a progesterone-receptor complex. Lastly, the treatment of chromatin with an antibody prepared against either single-stranded DNA or double-stranded DNA does not alter the extent of binding of the progesterone-receptor complex. Similarly, pretreatment of chromatin with a single-stranded nuclease does not inhibit the capacity of chromatin to bind the hormone-receptor complex.
Insights
Progesterone-receptor complex binding to chromatin differs between tissues primarily in the number of acceptor sites, not affinity. This binding is influenced by salt concentration and receptor activation, not DNA structure.
Area of Science:
- Molecular Biology
- Endocrinology
- Genetics
Background:
- Progesterone receptor complexes play a crucial role in gene regulation.
- Understanding the specifics of hormone-receptor binding to chromatin is key to deciphering cellular responses.
Purpose of the Study:
- To investigate the in vitro binding characteristics of progesterone-receptor complexes to chromatin from various tissues.
- To identify differences and similarities in binding affinity and site availability between target and nontarget tissues.
Main Methods:
- In vitro chromatin binding assays using progesterone-receptor complexes.
- Analysis of binding kinetics, including dissociation rates (K(D)) and acceptor site numbers.
- Experiments involving varying salt concentrations and DNA-modifying treatments.
Main Results:
- Progesterone-receptor complexes exhibit similar binding affinity (K(D) ≈ 3.10⁻⁹ M) to chromatin from both target and nontarget tissues.
- Significant differences were observed in the total number of acceptor sites, with oviduct chromatin having the most and erythrocyte chromatin the least.
- Binding is influenced by salt concentration, affecting receptor activation, and is independent of DNA single- or double-strandedness.
Conclusions:
- Tissue-specific differences in progesterone-receptor complex binding to chromatin are primarily determined by the number of available acceptor sites, not binding affinity.
- Salt concentration plays a critical role in modulating receptor activation and binding dynamics.
- The binding mechanism does not appear to be directly dependent on the structural state (single- or double-stranded) of DNA.