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Correlation of nuclear acceptor sites for oestrogen receptors with gene transcription in vitro.

ABSTRACT: The interaction of oestrogen receptors with their nuclear acceptor sites was studied to ascertain whether these acceptor sites are involved in the regulation of ovalbumin-gene expression in the chick oviduct. As previously described, two distinct oestrogen-receptor species exist, and both are translocated into the nucleus after oestrogen administration in vivo [Smith, Clarke, Zalta & Taylor (1979) J. Steroid Biochem.10, 31-35]. In the present investigation we observed that the tubular-gland-cell concentrations of cytoplasmic receptors (800-900/cell) do not vary with prolonged withdrawal, nor do the relative ratios of the two receptor types change; however, the nuclear accumulation and retention of these receptors after secondary oestrogen administration are attenuated in a time-dependent fashion. Chicks were withdrawn from oestrogen for 24-84h. Some animals were then restimulated with oestrogen and killed after 4h, when oviduct nuclei were isolated. These nuclei were assayed for nuclear receptor concentrations and for their capacity to synthesize ovalbumin mRNA in vitro. Although an equal number of cytoplasmic receptors appeared to be translocated, oestrogen-receptor occupancy within the nucleus was not equal, but was inversely proportional to the preceding length of withdrawal. This decrease in nuclear acceptor sites was accompanied by a similar decrease in the capacity of these same nuclei to transcribe ovalbumin mRNA in vitro. A statistical evaluation of nuclear oestrogen-receptor concentrations and ovalbumin-mRNA synthesis in vitro was made. Correlation analysis revealed a Pearson coefficient r=0.87 (P<0.001, n=17), indicating that a high degree of correlation exists between these two parameters. These results are consistent with the hypothesis that nuclear oestrogen-receptor-acceptor complexes may correspond to initiation sites for RNA polymerase II transcription of an oestrogen-regulated gene.

SUBMITTER: Taylor RN

PROVIDER: S-EPMC1162352 | biostudies-other | 1980 Nov

REPOSITORIES: biostudies-other

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Project description:Partially purified hen oviduct oestrogen receptors, charged with [3H]oestradiol, were shown to specifically bind in vitro to purified hen oviduct chromatin. Maximal binding occurred within 60min at 0 degrees C in a Tris buffer containing 0.1 M-KCl and 0.5 mM-phenylmethanesulphonyl fluoride. The binding of the [3H]oestradiol-receptor complexes to intact purified chromatin was saturable, whereas the receptor binding to hen DNA remained linear. Saturation was further demonstrated by the minimal acceptor binding of receptor charged with [3H]oestradiol plus 200-fold oestradiol compared with [3H]oestradiol receptors at equal [3H]oestradiol concentrations. Scatchard analysis of [3H]oestradiol-receptor binding to chromatin above DNA levels gave indications of high-affinity binding with a low capacity. Further, the nuclear binding was tissue-specific since the binding to hen spleen chromatin was negligible. To further uncover the specific acceptor sites, proteins were removed from hen oviduct chromatin by increasing concentrations of guanidine hydrochloride (1-7M). Those residual fractions extracted with 3-7 M-guanidine hydrochloride had the highest acceptor activity (above DNA levels) with the peak activity uncovered by 5 M-guanidine hydrochloride. To further characterize the oestrogen-receptor acceptor sites, oviduct chromatin was bound to hydroxyapatite in the presence of 3 M-NaCl and then protein fractions were extracted sequentially with 1-7 M-guanidine hydrochloride. Each fraction was then reconstituted to pure hen DNA by reverse gradient dialysis. [3H]Oestradiol receptors were found to bind to the greatest degree to the fraction reconstituted from the 5 M-guanidine hydrochloride protein extract. Reconstituted nucleoacidic proteins (NAP) from combined 4-7 M-guanidine hydrochloride protein extracts showed saturable binding by [3H]-oestradiol receptors, whereas binding to hen DNA did not saturate. The high affinity, low capacity, and specificity of binding of oestrogen receptors to NAP was similar to that found in intact chromatin. Thus, chromatin acceptor proteins for the oestrogen receptor have been partially isolated and characterized in the hen oviduct and display properties similar to that reported for the acceptor proteins of the progesterone receptor.

Project description:Endocrine signal transduction occurs through cascades that involve the action of both ligand-dependent and ligand-independent nuclear receptors. In insects, two such nuclear receptors are HR3 and E75 that interact to transduce signals initiated by ecdysteroids. We have cloned these nuclear receptors from the crustacean Daphnia pulex to assess their function as regulators of gene transcription in this ecologically and economically important group of organisms. Both nuclear receptors from D. pulex (DappuHR3 (group NR1F) and DappuE75 (group NR1D)) exhibit a high degree of sequence similarity to other NR1F and NR1D group members that is indicative of monomeric binding to the RORE (retinoid orphan receptor element). DappuE75 possesses key amino acid residues required for heme binding to the ligand-binding domain. Next, we developed a gene transcription reporter assay containing a luciferase reporter gene driven by the RORE. DappuHR3, but not DappuE75, activated transcription of the luciferase gene in this system. Co-transfection experiments revealed that DappuE75 suppressed DappuHR3-dependent luciferase transcription in a dose-dependent manner. Electrophoretic mobility shift assays confirmed that DappuHR3 bound to the RORE. However, we found no evidence that DappuE75 similarly bound to the response element. These experiments further demonstrated that DappuE75 prevented DappuHR3 from binding to the response element. In conclusion, DappuHR3 functions as a transcriptional activator of genes regulated by the RORE and DappuE75 is a negative regulator of this activity. DappuE75 does not suppress the action of DappuHR3 by occupying the response element but presumably interacts directly with the DappuHR3 protein. Taken together with the previous demonstration that daphnid HR3 is highly induced by 20-hydroxyecdysone, these results support the premise that HR3 is a major component of ecdysteroid signaling in some crustaceans and is under the negative regulatory control of E75.

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Correlation of nuclear acceptor sites for oestrogen receptors with gene transcription in vitro.

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