Digestion of chromosomal proteins in formaldehyde treated chromatin

Treatment of chromatin subunits (nucleosome monomers) with formaldehyde results in the formation of cross-links between DNA and histones and between histones and histones. Digestion of chromosomal proteins with proteinase K does not lower the protein/DNA weight ratio below 0.08 to 0.1 as determined by cesium chloride gradient centrifugation of the digestion product from formaldehyde-treated nucleosomes. In addition to proteinase K, formaldehyde-treated nucleosomes were tested for accessibility to trypsin and pronase. The CsCl gradient patterns show, that pronase digestion and proteinase K treatment yield similar results. Trypsin treatment of control and formaldehyde-treated nucleosomes shows, that the sites which are accessible for trypsin in native nucleosomes, are blocked after formaldehyde treatment. Analysis of the CsCl gradient peak fractions in polyacrylamide gels shows, that the reliability of DNA fragment size determinations depends on the completeness of deproteinization.     

Effect of hypophysectomy on liver nuclear ribonucleic acid synthesis in aging rats.

Changes in RNA synthesis in liver nuclei were observed at different ages and after hypophysectomy and hormone replacement in female Sprague-Dawley rats. As determined by the incorporation of [3H]UMP into an acid-insoluble product, RNA synthesis decreased by about 75% in intact rats from 6 months to 24 months of age. This decline with age was not observed in liver nuclei from 24-month-old rats that had been hypophysectomized at 12 months and maintained on a minimal hormone-replacement therapy. Thyroid hormones and somatotropin (growth hormone) had an additive effect on RNA synthesis in liver nuclei from these hypophysectomized rats. The same hormones had no significant effect on intact, age-matched rats. With advancing age, nuclei of intact rats had an increase in the pool of free RNA polymerase and an apparent decrease in the enzyme activity bound to nuclear chromatin. There was no change in total enzyme with age. In hypophysectomized, hormone-treated rats, free RNA polymerase activity decreased and chromatin-bound activity increased. There was no difference in total nuclear RNA polymerase activity between operated or intact rats. However, the ratio of the bound to the free activity was different. These results suggest that the ability of RNA polymerase to bind to chromatin may be involved in the age-related decrease in liver nuclear RNA synthesis of intact rats.     

Denaturation of deoxyribonucleic acid in situ effect of formaldehyde.

In situ denaturation of nuclear deoxyribonucleic acid (DNA) is studied by use of acridine orange to differentially stain native versus denatured DNA, and a flow-through cytofluorometer for measurements of cell fluorescence. Thermal- or acid-induced DNA denaturation is markedly influenced by formaldehyde. Two mechanisms of the formaldehyde action are distinguished. If cells are exposed to the agent during heating, DNA denaturation is facilitated, most likely by the direct action of formaldehyde as a "passive" denaturing agent on DNA. If cells are pretreated with formaldehyde which is then removed, DNA resistance to denaturation increases, presumably due to chromatin cross-linking. It is believed that both effects occur simultaneously in conventional techniques employing formaldehyde to study DNA in situ, and that the extent of each varies with the temperature and cell type (chromatin condensation). Thus, profiles of DNA denaturation of cells heated with formaldehyde do not represent characteristics of DNA denaturation in situ; DNA denaturation under these conditions is modulated by the reactivity of chromatin components with formaldehyde rather than by DNA interactions with the macromolecules of nuclear mileu.     

Hybridization selection of covalent nucleic acid-protein complexes. 2. Cross-linking of proteins to specific Escherichia coli mRNAs and DNA sequences by formaldehyde treatment of intact cells

Proteins cross-linked to pBR322 mRNAs and DNA by formaldehyde treatment of intact Escherichia coli cells have been detected with the use of a novel detection method. Among the proteins cross-linked to pBR322 mRNAs were S1, S21, and at least six other proteins of the small ribosomal subunit, initiation factor 1, elongation factor (EF) Tu, and very small amounts of EF-G and EF-Ts. The single strand binding protein, the HU-proteins, and RNA polymerase subunits alpha and beta were among the proteins cross-linked to pBR322 DNA. The results obtained suggest that the procedures described, can also be used to study interactions between different nucleic acid-bound polypeptides. The results are discussed in relation to the working mechanism of formaldehyde, and are compared to the results obtained with cross-linking induced by ultraviolet light. The methods presented should also be of use for the study of nucleic acid-protein interactions in other organisms.     

Conformational changes in E. coli RNA polymerase during promoter recognition.

We analysed complexes formed during recognition of the lacUV5 promoter by E. coli RNA polymerase using formaldehyde as a DNA-protein and protein-protein cross-linking reagent. Most of the cross-linked complexes specific for the open complex (RPO) contain the beta' subunit of RNA polymerase cross-linked with promoter DNA in the regions: -50 to -49; -5 to -10; + 5 to +8 and +18 to +21. The protein-protein cross-linking pattern of contacting subunits is the same for the RNA polymerase in solution and in RPO: there are strong sigma-beta' and beta-beta' interactions. In contrast, only beta-beta' cross-links were detected in the closed (RPC) and intermediate (RPI) complexes. In presence of lac repressor before or after formation of the RPO cross-linking pattern is similar with that of RPI (RPC) complex.     

Possible function of RNA-binding proteins of eukaryotes: blocking of RNA accessibility for DNA-interacting (regulatory) protein

In the presence of rat liver cytosol the glucocorticoid receptor complexes (GRC) bind very weakly to RNA as compared to DNA, whereas the binding of purified GRC to RNA is only 2-3 time less than to DNA. It is assumed that the RNA-binding proteins present in the cytoplasm selectively prevent the GRC binding to RNA and thus facilitate the GRC binding to nuclear DNA (chromatin). This blocking function may be performed by the RNA-binding proteins with respect to different regulatory DNA-dependent intracellular proteins in vivo. Possible mechanisms of regulation of the genome activity based on changes in RNA-RNA-binding proteins ratio in intact cells and in individual cell compartments are discussed.     

Nuclear location of hormone-free estrogen receptors by monoclonal antibodies could be a tissue-fixation dependent artifact

The 'two-step' model proposed by Jensen and his collaborators for explaining estrogen action conceptualized hormone-free estrogen receptors (ER) to be cytoplasmic, and hormone-filled, transformed ER to be nuclear. Applying monoclonal antibodies which recognized epitopes in ER and formaldehyde-fixed tissues, King et al demonstrated exclusively nuclear staining in target tissues utilizing immunoperoxidase technique. Recently these antibodies have become commercially available enabling other investigators to conduct studies. In this report, using these monoclonal antibodies we have demonstrated that a change in the concentration of formaldehyde alters the staining pattern yielding cytoplasmic instead of nuclear staining in calf uterus, MCF-7 cells, and ER(+) human breast cancer. In addition, neutralization of the antibody activity was not achieved with freshly prepared ER(+) cytosols. Formaldehyde-treated cytosols were essential. These results ought to caution investigators in determining in vivo location of antigens based on the staining pattern obtained in fixed tissues. Furthermore, this effect of formaldehyde on estrogen receptors may be applicable to other steroid hormone receptors.     

Stimulation of ribonucleic acid polymerase activity in vitro by prostatic steroid–protein receptor complexes

A system has been developed which allows the stimulation in vitro of prostatic RNA polymerase by prostatic 5alpha-dihydrotestosterone-protein receptor complexes prepared from the tissues of castrated rats. The reconstitution in vitro of such a system necessitates the purification of several subcellular components. Two 5alpha-dihydrotestosterone-receptor complexes are located in the prostatic soluble supernatant fraction, separable by selective ammonium sulphate fractionation, and one complex can be isolated from the nuclear fraction. In the presence of all these complexes, stimulation of RNA polymerase in intact nuclei and nucleoli was observed. The complexes also increased the activity of the enzyme solubilized from whole nuclei. Greater stimulation of this system was noted in the presence of prostatic chromatin as template, as compared with that observed with calf thymus DNA or liver chromatin as template. The effects of the complexes on subnuclear forms of RNA polymerase, of nucleolar and extranucleolar origin, are also described. RNA polymerase solubilized from nucleoli is more susceptible to stimulation by the 5alpha-dihydrotestosterone-receptor complexes than is the ;nucleoplasmic' enzyme. Stimulation occurs less readily in the presence of Mn(2+) and at high ionic strength than in the presence of Mg(2+) and at low ionic strength. Preliminary experiments show that prostatic nucleolar RNA polymerase transcribes prostatic chromatin poorly as compared with the nucleoplasmic enzyme. The observations reported indicate an involvement of non-histone proteins associated with DNA in the process by which stimulation of enzyme activity by the 5alpha-dihydrotestosterone-receptor complexes is achieved. The implications of these findings in the mechanism of steroid hormone action is considered.     

Nuclear thyroid hormone receptors: evidence for association with nucleolar chromatin.

When hypothyroid rat liver nuclei labeled $\text{in vivo}$ with [125 I]L-triiodothyronine are incubated with micrococcal nuclease, the nuclear chromatin is digested and chromatin particles are released into the medium. The nuclease-treated nuclei contain intact nucleoli and a residual chromatin fraction. When this residual chromatin is purified, it contains only a small percentage of the initial nuclear DNA but is strikingly enriched in [125 I]L-triiodothyronine. This chromatin fraction has many of the characteristics of nucleolar chromatin including a high protein to DNA ratio, an abundance of nonhistone proteins, and a relatively high RNA to DNA ratio. An association of thyroid hormone receptors with a nucleolar component implicates this organelle in the early events of thyroid hormone action.     

Nature of functional estrogen receptors at the plasma membrane

Although rapid signaling by estrogen at the plasma membrane is established, it is controversial as to the nature of the receptor protein. Estrogen may bind membrane proteins comparable to classical nuclear estrogen receptors (ERs), but some studies identify nonclassical receptors, such as G protein-coupled receptor (GPR)30. We took several approaches to define membrane-localized estrogen-binding proteins. In endothelial cells (ECs) from ERalpha/ERbeta combined-deleted mice, estradiol (E2) failed to specifically bind, and did not activate cAMP, ERK, or phosphatidyinositol 3-kinase or stimulate DNA synthesis. This is in contrast to wild-type ECs, indicating the lack of any functional estrogen-binding proteins in ERalpha/ERbeta combined-deleted ECs. To directly determine the identity of membrane and nuclear-localized ER, we isolated subcellular receptor pools from MCF7 cells. Putative ER proteins were trypsin digested and subjected to tandem array mass spectrometry. The output analysis identified membrane and nuclear E2-binding proteins as classical human ERalpha. We also determined whether GPR30 plays any role in E2 rapid actions. MCF7 (ER and GPR30 positive) and SKBR-3 (ER negative, GPR30 positive) cells were incubated with E2. Only MCF7 responded with significantly increased signaling. In MCF7, the response to E2 was not different in cells transfected with small interfering RNA to green fluorescent protein or GPR30. In contrast, interfering RNA to ERalpha or ER inhibition prevented rapid signaling and resulting biology in MCF7. In breast cancer and ECs, nuclear and membrane ERs are the same proteins. Furthermore, classical ERs mediate rapid signals induced by E2 in these cells.     

Growth hormone and drug metabolism. Acute effects on nuclear ribonucleic acid polymerase activity and chromatin.

Adult male rats, subjected either to sham operation or to hypophysectomy and adrenalectomy were maintained for 10 days before treatment with growth hormone. Results of the acute effects of growth hormone on the rat liver nuclear RNA polymerase I (nucleolar) and II (nucleoplasmic) activities as well as the chromatin template capacity were then studied and compared with the growth-hormone effects on the drug metabolism described in the preceding paper (Wilson & Spelsberg, 1976). 2. Conditions for isolation and storage of nuclei for maintenance of optimal polymerase activities are described. It is verified that the assays for polymerase activities require a DNA template, all four nucleoside triphosphates, and a bivalent cation, and that the acid-insoluble radioactive product represents RNA. Proof is presented that under high-salt conditions DNA-like RNA (polymerase II) is synthesized, and that under low-salt conditions in the presence of alpha-amanitin, rRNA (polymerase I) is synthesized. 3. In the livers of hypophysectomized/adrenalectomized rats, growth hormone increases the activity of both RNA polymerase enzymes and the chromatin template capacity within 1h after treatment. The effects last for 12h in the case of polymerase II but for only 6h in the case of polymerase I. Sham-operated rats respond to growth hormone in a manner somewhat similar to that shown by hypophysectomized/adrenalectomized rats. These results, which demonstrate an enhancement of RNA polymerase I activity in response to growth hormone, support those from other laboratories. 4. Growth-hormone enhancement of the chromatin template capacity in the liver of hypophysectomized/adrenalectomized rats contrasts with previous reports. The growth-hormone-induced de-repression of the chromatin DNA could represent the basis of the growth-hormone-induced enhancement of RNA polymerase II activity in the hypophysectomized/adrenalectomized rats, although some effect of growth-hormone on the polymerase enzymes is still suggested.