"Bioassays of Isolated Nuclear RNA Species as Activators of DNA Transcription".*

Jeannette A. Hovsepian ^{1, @} and John H. Frenster ^{2, @}

Departments of  ¹Radiology and of  ² Medicine, Stanford University School of Medicine, Stanford, California 94305, USA

^@ Present Address:  Physicians' Educational Series, Atherton, California, 94027-5446 USA
Phone:  +1 650 367 6483;   Fax:   +1 650 364 1773
e-mail:   frenster@euchromatin.net

* Supported in part by a USPHS Research Career Development Award (CA-17857) from the National Cancer Institute to J.H.F., and by USPHS Research Grants from the National Cancer Institute.

Active DNA transcription is confined to the euchromatin portion of the mammalian cell nucleus in vivo, and this activity is preserved within the euchromatin fraction after its gentle isolation from the nuclei. Similarly, repressed DNA transcription is found within the heterochromatin portion of the cell nucleus in vivo, and this repression is preserved within the heterochromatin fraction after its gentle isolation from the nuclei. Isolated euchromatin and heterochromatin fractions can then be used as parallel bioassay systems for molecular species that are capable of either repressing or activating DNA transcription in vitro in a nucleus-free system. Addition of separate species of polyanions, each isolated from nuclei, to incubations of isolated heterochromatin result in varying degrees of activation of DNA transcription within such isolated heterochromatin. The most active of such nuclear polyanions include small RNA molecules that are stable within the cell nucleus, and which return to the cell nucleus after cycling to the cytoplasm during cell mitosis (Goldstein L, (1974), Exp. Cell Res. 89: 421-425). Such RNA species are active in bioassays in the absence of added protein, and are found to a lesser degree after extraction from total cytoplasm, from nuclear ribosome fractions, from total yeast, or from total bacterium sources. Isolated RNA species, positive as activators of DNA transcription within isolated repressed heterochromatin bio-assays, have no further effect on already-active isolated euchromatin in parallel bio-assay incubations. These bioassay data are compatible with biochemical analyses which reveal that isolated euchromatin fractions contain a 5-fold excess of RNA species when compared to isolated heterochromatin fractions. 

In assays for cell biology, concern for in vivo structure encompasses many restraints; not only must the covalent bonds of molecular sequences and groups be preserved, but also the hydrogen bonds and the hydrophobic forces of interacting motifs must be gently preserved, for the assays to be meaningful for interpreting the in vivo  state [v1].

Only under extremely gentle conditions can such diverse cellular processes as RNA synthesis, DNA synthesis, or protein synthesis be observed in the cell-free state, much less in the nuclear-free or cytoplasm-free state. When considering RNA synthesis within isolated chromatin complexes, care must be given to choosing the active versus the repressed chromatin as a sample, depending upon which function is being studied, either the details of the existing active state within the cell, or the ability of the existing repressed state to become activated by in vivo activators in vitro [v2].

Fig. 1a. Swollen nuclei displaying condensed masses of repressed chromatin and extended 10 nm microfibrils of active chromatin (x 16,875), [4].

Higher magnifications reveal the active chromatin microfibrils to be of 10 nm diameter (Fig. 1b), and these microfibrils can be traced for up to 1.0 um. of their length (Fig. 1b).

Fig. 1b. Detail of swollen nucleus displaying the structural continuity of active 10 nm microfibrils with repressed masses of chromatin (x 45,000), [4].

These extended 10 nm microfibrils of active chromatin are seen to be structurally continuous with a dense reticulum of fibers within the condensed masses of repressed chromatin (Fig. 1b). The zone of transition between the extended 10 nm microfibrils and the condensed masses is sharp, occurring within less than 10 nm of the length of the microfibrils.

It has become possible to isolate from calf thymus lymphocytes both the active euchromatin fraction and the repressed heterochromatin fraction from the same animal sample, and to use these two isolated chromatin fractions as the basis for the assay of activating factors and repressing factors for an in-vitro RNA synthesis which reflects the in-vivo state. Such isolation techniques involve working in a cold room at 4^oC., using only isotonic solutions that preserve a stable pH, selecting Mg²⁺ and Mn²⁺ concentrations ideal for subsequent metabolic incubations, and avoiding tissue denaturation or trauma, [3].

Methods:

The nuclear polyanion contents of active and repressed chromatin fractions prepared from the same animal were determined relative to the DNA contents, active chromatin was found to contain a two-fold excess of total non-histone residual proteins remaining after extractions of histones, a five-fold excess of total RNA and of total phospholipids, and an almost four-fold excess of total phosphoprotein phosphorus. Such nuclear phospho-proteins constitute up to 15 percent of the non-histone residual proteins. Previous studies have suggested the importance of the non-histone residual proteins in the metabolism of chromatin and within certain neoplasms. A parallel increase in both non-histone proteins and in RNA has recently been found in the soluble chromatin of active tissues in the chicken, in the active puffs of polytene chromosomes, and in the active loops of lampbrush chromosomes. The presence of these nuclear polyanions in excess within active chromatin suggests that they may play a part in antagonizing the DNA-histone interaction within active chromatin, [5].

Active and repressed chromatin fractions were isolated from the same animal and washed once in 0.25 M sucrose-0.010 M MnCl₂ to avoid subsequent gelation, the synthesis of RNA was then studied in the isolated state. In such experiments, the chromatin fractions contained both the RNA polymerase and the template DNA needed for such synthesis, and were suspended as equal concentrations of either active chromatin or repressed chromatin (4-8 mg. DNA/ml.) from the same animal in an incubation medium of the following composition: 0.1 M tris-hydroxymethy-aminomethane HCl, pH 7.7, 0.005 M MgCl₂, 0.002 M MnCl₂, 0.04 M NaF, 0.002 M GTP, CTP, and either ATP or UTP, in an incubation volume of 2.0 ml./flask, to which was added 0.5 uC. uridine-2-¹⁴C-triphosphate (spec. act. 21.0 mC./mM) or 0.5 uC. adenosine -8-¹⁴C-triphosphate (spec.act. 30.9 mC./mM). The flasks were shaken in air at 37^o C. for up to 30 min., the reaction was stopped by the addition of 5.0 ml. of ice-cold 10 percent trichloroacetic acid, and the DNA, RNA, protein, and incorporated radioactivity determined as previously described, [5].

Results:

After isolation of active euchromatin and repressed heterochromatin fractions, detailed biochemical, biophysical, and metabolic studies were possible on the isolated fractions prepared in parallel from the same animal, [5].

Fig. 1c. Histone proteins stabilize DNA molecules against thermal melting within both isolated active euchromatin and isolated repressed heterochromatin, with a greater effect noted within repressed heterochromatin. The lower median melting temperature found within isolated euchromatin compared to isolated heterochromatin suggests a partial DNA melted state within isolated euchromatin, [5].

Table 1. Relation of Nuclear Constituents to DNA within Isolated Chromatin Fractions.

	No. of Animals	Active Chromatin (mg/100 mg DNA)	Repressed Chromatin (mg/100 mg DNA)	Active/repressed
Total histones	10	90.7 +/- 7.7	101.1 +/- 9.1	0.90 +/- 0.07
Non-histone  residual proteins	4	109.0 +/- 6.1	54.9 +/- 5.7	2.00 +/- 0.08
Total  phospholipids	5	17.0 +/- 3.6	3.7 +/- 1.3	4.93 +/- 1.26
Total ribonucleic acids	8	9.0 +/- 4.7	1.8 +/- 0.7	5.15 +/- 2.86
Total phospho- protein phosphorous	4	0.418 +/- 0.028	0.117 +/- 0.019	3.74 +/- 0.56
Mean +/- S.E.

Table 1. Cationic histones react with the anionic phosphate groups of the DNA molecule, and are equally distributed between active euchromatin and repressed heterochromatin. By contrast, nuclear polyanions such as RNA, phosphoproteins, non-histone residual proteins and phospholipids are found in excess within active euchromatin. RNA has the highest ratio of such preferential binding to active euchromatin, [5].
 

Table 2. Trypsin preferentially digests histone proteins, releasing repression in both already-active euchromatin and in repressed heterochromatin to levels of RNA synthetic activity above control values, [5].

Table 3. The addition of isolated single nuclear polyanions has little effect on the RNA synthetic activity of already-active isolated euchromatin, but does increase the RNA synthetic activity of isolated repressed heterochromatin. The addition of total nuclear RNA is most effective in activating repressed isolated heterochromatin. The addition of polycationic histone proteins is repressive when added to either isolated chromatin fraction, [5].

Figure 2. Isolated active euchromatin and repressed heterochromatin retain the differential RNA synthetic activity after isolation that they displayed within the cell nucleus before chromatin fractionation, [5].

Figure 3. Polyethylene sulfonate is a very strong synthetic polyanion, and its addition to the incubation has marked effects on the RNA synthetic activity of both isolated active euchromatin and isolated repressed heterochromatin. Polyethylene sulfonate captures histone protein molecules from both isolated chromatin fractions.

By contrast, the addition of thymus total RNA stimulates RNA synthetic activity more moderately, by displacing histone proteins from the DNA molecule, and has little effect on already-active isolated euchromatin.

In further contrast, the addition of total yeast RNA is actually inhibitory to RNA synthetic activity of already-active euchromatin, only becoming stimulatory at higher dose levels, [5].

Figure 4. Added RNA species vary in their effects on the RNA synthetic activity of isolated repressed heterochromatin. Thymus nuclear RNA is most active, while thymus cytoplasmic RNA, yeast RNA and E. coli S-RNA are less active. Thymus nuclear ribosomal RNA is least active. All added RNA species were isolated free of protein, [5].

 Fig. 5: Left, polyelectrolyte interaction during repression and de-repression. Polycationic protein repressors form electrostatic complexes with the negative phosphate groups of both strands of the DNA double helix, inhibiting DNA strand separation during RNA synthesis. Such protein repressors can be partially displaced from portions of the DNA genome either by trypsin hydrolysis or by the electrostatic antagonism of such polyanions as polyethylene sulfonate, phosphoproteins, or nuclear de-repressor RNA, [v2].

Fig. 5: Right, specificity of de-repression within the active euchromatin complex. Repressor proteins are partially displaced from portions of the DNA genome by nuclear polyanions, allowing intermittent strand separation of the DNA double helix. Specific de-repressor RNA hybridizes with a single DNA strand, freeing the complementary DNA strand for continuous pre-messenger RNA synthesis, [6].

Conclusions:

1. Gently-prepared fractions of active euchromatin and repressed heterochromatin can be isolated from the same sample of isolated mammalian nuclei, and retain their characteristic activity for or against RNA synthesis in subsequent metabolic incubations of each fraction.

2. Isolated active euchromatin 10 nm microfibrils contain 3-5x as many nuclear polyanions as does isolated repressed heterochromatin.

3. When these polyanions are added to incubations of isolated repressed heterochromatin, the rate of RNA synthesis is activated to near that of isolated active euchromatin.

4. Among these activating polyanions, the most active are species of nuclear RNA.

5. Activating nuclear RNA has little additional effect on already-active euchromatin.

6. Activating RNA from yeast is initially inhibitory on already-active mammalian euchromatin.

7. At higher concentrations of yeast RNA, additional activation is seen to a base line of untreated
already-active mammalian chromatin.

8. These yeast RNA mechanisms suggest a simple RNA-displacement and RNA-replacement mechanism during re-programming of islolated already-active mammalian euchromatin, [18].
 

References:

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15a. Frenster JH, "Nuclear RNA Species Activate DNA Transcription Within Chromatin", FASEB Journal, Vol. 13, No. 7, A1506 (April 23, 1999).

15b. Frenster JH, "Oncogenes as Molecular Targets within Active Chromatin", Clinical Cancer Research, vol. 5, suppl. l, p. 3855s, (624), (November, 1999).

16. Frenster JH, "Nuclear Ribosomes and RNA-RNA Duplexes", Mol. Biol. Cell vol. 11, suppl. p. 20a (December, 2000).

17. Frenster JH, "Activation of DNA Transcription within Repressed Chromatin by Nuclear RNA Species", RNA 2001, p. 237 (The RNA Society, Bethesda, Maryland, 2001).

18. Frenster JH, "Yeast  RNA  Re-Programming  of  Already-Active  Mammalian Chromatin", RNA 2002, p. 592, (2002, Bethesda, MD: The RNA Society).

19. Frenster JH, "Uni-Polar Clustering of Lymphocyte DNA Templates Toward Neoplastic Target Cells Within Hodgkin’s Disease Lymph Nodes", Proc. Am. Assoc. Cancer Res. vol. 43, p. 1134 (March, 2002).

20. Frenster JH, and Hovsepian JA, "RNA Feedback Mechanisms during Eukaryotic Gene Regulation", Biomolecular Systems Initiative, "Northwest Symposium on Systems Biology", p. 15, Oct. 17-18, 2002.

21. Hovsepian JA, and Frenster JH, "RNA-Induced Melting of DNA during Selective DNA Transcription", Molec. Biol. Cell, vol. 13, supp. p. 239a (November, 2002).

22. Frenster JH, and Hovsepian JA, "Overshoot in Late Telophase for RNA Re-Programming of Mitotic Chromatin", "RNA2003", p. 211 (2003), The RNA Society, Bethesda, MD, USA.

23. Hovsepian JA, and Frenster JH, "Euchromatin as an Extensile Force within Mammalian Cell Nuclei", Molec. Biol. Cell, vol. 14, supp. p. 93a (November, 2003).

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