John H. Frenster
Department of Medicine
Stanford University School of Medicine
Stanford, California 94305

Summary.
Introduction.
Selective Transcription.
   Chart 1: Diversity of effects on isolated chromatin.
DNA Helix Openings.
   Table 1: Correlations of nuclear ligand binding and the effects on RNA synthesis.
Histone Displacement.
Acidic Chromatin Macromolecules.
De-Repressor RNA.
   Chart 2: Model of selective gene de-repression by RNA.
   Chart 3: Feedback effects of excessive RNA synthesis.
De-Repression by Exogenous DNA.
   Chart 4: De-repressive effects of oncogenic viral DNA.
Helix Openings in Supercoils.
Aging, Oncogenesis, Radiobiology, and Ultrastructure.
   Chart 5: Gene de-repression effects within human neoplasms.
Support.
References.
Addendum.
Additional Reference.
Links. 

DNA helices must undergo openings in the form of localized strand separations in order to permit the onset of RNA synthesis or DNA synthesis. The selective control of such DNA helix openings at particular gene loci is the critical feature of gene regulation in prokaryotes and eukaryotes.

Introduction:

Gene regulation implies differential activity of DNA molecules within an individual cell. Such differential activity is observed during both gene transcription and gene replication (25), and is a feature of DNA molecules within both prokaryotes (10) and eukaryotes (30). Because gene replication appears to be secondary to gene transcription (11, 45), the critical feature of the regulatory event appears to lie in the molecular details of selective gene transcription (10, 23). Such selectivity involves both the choice of a particular gene locus and the choice of a particular DNA strand on which to effect mRNA synthesis (25).

Selective Transcription:

Each normal diploid cell of an individual animal contains a full and identical complement of all DNA molecules characterizing that animal (46, 51). Only a limited number of DNA molecules are transcribed to RNA within any one cell or tissue (29), and these are characteristic of the particular tissue type (46, 58).

Stable epigenetic mechanisms select specific portions of the genome for transcription (15, 36), and these mechanisms can be defined within isolated native complexes of repressed heterochromatin and de-repressed euchromatin prepared in parallel from a single tissue sample (29). By these analyses, polycationic histone proteins (Chart 1) are found to combine nonspecifically with the phosphate groups on the exterior of the DNA helix (21, 56). 

The effect of such histone molecules is to stabilize the DNA helix against the helix openings that follow localized separations of the DNA strands (21, 23), and, concurrently, to decrease markedly the template activity of the DNA molecule (28, 40). This concordence of effects in fact provided the first evidence that DNA helix openings via localized strand separations are a necessary molecular feature of DNA molecules during the RNA synthesis of selective gene transcription (22, 23).

DNA Helix Openings:

Recently, many additional examples of localized DNA helix openings have been recognized in both bacterial (12, 16) and mammalian (13, 61) cells. Nuclear ligands other than histones that share with histones the ability to bind preferentially to double-stranded DNA, 

A great variety of molecules with biological activity penetrate into the cell nucleus and bind to DNA. These nuclear ligands can be classified as to their preference for binding to DNA in the double-stranded state or in the single-stranded state (24). Those ligands binding preferentially to DNA in the double-stranded state are all found to decrease the rate of RNA synthesis by DNA after such binding. Conversely, those ligands binding preferentially to DNA in the single-stranded are all found to increase the rate of RNA synthesis by such DNA after binding (24). These data are consistent with a ligand-induced closing or opening of DNA helices, respectively, and indicate the central role played by such DNA helix openings in the molecular control of selective transcription (24).

and are thereby capable of stabilizing the DNA helix against helix openings, have similarly been found to decrease the template activity of DNA molecules (24), thus further confirming the requirement for helix openings in DNA template molecules during the RNA synthesis of selective gene transcription (23, 24).

Conversely, nuclear ligands capable of binding preferentially to single-stranded DNA (24), and thereby capable of inducing DNA helix openings (24) have been found to be capable also of increasing the activity of DNA templates for RNA synthesis, again indicating the crucial role of helix openings for DNA template activity in selective transcription (24). It is now possible to analyze separately both the equilibrium and kinetic aspects of the interaction of such DNA ligands with the DNA molecule (52).

Histone Displacement:

In animal cells, histones are usually in close apposition with underlying DNA templates and therefore must be displaced from such templates before gene transcription can occur (22, 23). Many examples of such displacement of histones from active DNA template sites have now been demonstrated, including histone displacement within isolated de-repressed euchromatin complexes (21), within lymphocytes undergoing activation (44), within activated cell hybrids (6), within the atypical lymphocytes of infectious mononucleosis (5), and with the entrance of inactive mitotic cells into active interphase (54).

Conversely, evidence for the repositioning of displaced histones during inactivation of the DNA template has also been found within a variety of animal systems, including within the cells progressing through spermatogenesis (34), within cells inactivated by high-density culture (67), and within cells progressing through normal bone marrow cell differentiation and early mitosis (54). In each of these cell systems, histone displacement is correlated with DNA template activity for RNA synthesis, while histone repositioning is correlated with DNA template inactivity for RNA synthesis (31).

Acidic Chromatin Macromolecules:

The molecular mechanisms mediating histone displacement and de-repression are complex and usually involve a variety of acidic macromolecules found in association with DNA in de-repressed euchromatin complexes (21). These acidic macromolecules include acidic nonhistone residual proteins, phosphoproteins, lipoproteins, and RNA, all of which are capable of displacing histones from DNA templates, thereby converting repressed heterochromatin to de-repressed euchromatin, with a resultant marked increase in the rate of RNA synthesis (21, 39). The tissue-specific and gene-selective properties of gene transcription are mediated by macromolecular species contained in these acidic chromatin supernatants (2-4, 32, 41). The preparative methods for obtaining these acidic macromolecules all include significant amounts of nRNA (9, 17, 21, 32, 49, 65, 66), and it has been shown that such nRNA can de-repress previously repressed heterochromatin, markedly increasing the rate of RNA synthesis (21), and conferring both tissue and gene selectively upon the transcription process (3, 4, 14, 41).

De-repressor RNA:

These species of nRNA, variously referred to as de-repressor RNA (22), chromosomal RNA (3, 4, 38), or activator RNA (8), are capable of hybridizing to double-stranded DNA in 5 M urea solutions (3, 4, 41), apparently binding to the non-coding strand of DNA at a particular gene locus, thereby forming a DNA helix opening (Chart 2);

Chart 2. Derepressor RNA (chromosomal RNA, activator RNA) has the structural capability for selecting particular gene loci by combining with the complementary sequences on the anticoding strand of the DNA helix opening. This frees the remaining strand for single-strand specific mRNA synthesis (22). The rigidity imposed by the histones along the length of the DNA helix serves to concentrate any torsional influences on the helix to the area of histone displacement, where such torsion can result in induced DNA strand separations and the formation of a DNA helix opening.

and freeing the remaining coding strand of DNA for mRNA synthesis (8, 22).

As RNA synthesis proceeds, the immediate transcription product consists of both repeated and unique sequences (38), and appears to be capable of forming partially-double-stranded heterometric duplex RNA 

Chart 3. The immediate transcription product RNA consists of both repetitive and single-copy sequences on the same high-molecular weight molecule (37), corresponding to operator gene (o) and structural gene (sg) sequences (8, 30). Operator RNA (oRNA) is complementary in base composition to derepressor RNA (dRNA) and is capable of forming heterometric or homometric RNA-RNA duplexes with derepressor RNA after excessive rates of gene transcription, thereby inducing the removal of derepressor RNA from the DNA helix opening and providing a gene-specific mechanism for feedback inhibition of RNA synthesis (30).

or fully double-stranded homometric duplex RNS (47, 53, 60) with the 5' end of the RNA molecule (42), probably representing hybrids between de-repressor RNA and the operator RNA of the immediate transcription product (30). Such de-repressor or chromosomal RNA has been shown to be of moderately repetitive sequence composition (38, 59) and may be bound by the operator RNA sequences of the immediate transcription product when transcription rates at a particular gene locus are excessive, resulting in removal of de-repressor RNA from the DNA template, closure of the DNA helix opening, and an effective feedback inhibition of gene transcription specific for individual gene loci (30). De-repressor RNA may also represent that nRNA that migrates to the cytoplasm during early mitosis and returns to the nucleus early in interphase when RNA synthesis is resuming (35).

The remaining acidic macromolecules of the chromatin supernatant may also de-repress previously repressed heterochromatin and increase the rate of RNA synthesis (21, 32), but these are thought not to be the active molecules in selective gene transcription but rather consequences of such transcription (17, 58).

De-repression by Exogenous DNA.

A variety of DNA molecules can also interact with specific portions of the cellular genome, thereby inducing DNA helix openings and increasing the rate of RNA synthesis at the particular gene locus. 

Transformation by bacterial DNA of competent target bacterial cells involves interactions of single-stranded transforming DNA with the DNA of the host genome (18). Similarly, transformation by exogenous DNA in eukaryotic systems is thought to involve a single-stranded interaction with the host genome (19, 48).

Finally, oncogenic viral DNA undergoes single-stranded integration with the host genome (26, 27, 62), thereby forming a DNA helix opening and allowing both mRNA synthesis on the remaining free host DNA strand and anti-mRNA synthesis on the remaining free viral DNA strand. mRNA and anti-mRNA may then form double-stranded RNA duplexes in viral-transformed cells (1), while single-strand breaks in the integrated DNA sequences can free the viral genome (50, 64) and result in reversion of the transformed cell to normal phenotypic behavior (55). A similar reversion can be observed after removal of the viral genome by a variety of polar compounds (63).

Helix Openings in Supercoils:

The recent findings concerning the quarternary structure of chromatin (7) suggest that superhelical coils of DNA helices may favor DNA helix openings in DNA segments from which covering histones have been displaced, in a manner similar to the effects of such superhelical twisting on DNA helix openings in covalently closed circular bacterial and viral DNA (10, 11). In this instance, the presence of histones on intervening segments of DNA may accentuate the degree of local DNA helix opening at the target site by preventing it any other site.

Aging, Oncogenesis, Radiobiology, and Ultrastructure:

It will obviously be important to explore the consequences of DNA helix openings and their control during aging (13), oncogenesis (39) (Charts 4 and 5), and radiation (43), as well as in the finer details of chromatin fibril ultrastructure (20). 

Support:

Supported in part by Research Grants CA-10174 and CA-13524 from the National Cancer Institute, by Research Grant IC-45 from the American Cancer Society, and by a Research Scholar Award from the Leukemia Society.

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Since this review was completed in 1975, additional data have been published in providing experimental details of the role of DNA helix openings and nuclear RNA in gene regulation (1, 3-5, 7), carcinogenesis (6, 8), and cell activation (2):

1. Darzynkiewicz Z, Traganos F, Sharpless T, and Melamed MR, "Thermal Denaturation of DNA in situ as Studied by Acridine Orange Staining and Automated Cytofluorometry", Exptl. Cell Res. 90: 411-428 (1975).

2. Frenster JH, "Phytohemagglutinin-Activated Autochthonous Lymphocytes for Systemic Immunotherapy of Human Neoplasms", Ann. N.Y. Acad. Sci.: 277: 45-51 (1976).

3. Frenster JH, Landrum SR, Masek MA, and Nakatsu SL, "Control of DNA Helix Openings during Gene Regulation", Biophys. J. 16: 225a (1976).

4. Frenster JH, Landrum SR, Masek MA, Nakatsu SL, and Wilson LS, "Control of DNA Helix Openings During In-Vivo Normal and Neoplastic Cell Maturation", in: Fishman WH, and Sell S, (eds.), "Onco-Developmental Gene Expression", pp. 107-114, New York: Academic Press, Inc. (1976).

5. Frenster JH, Landrum SR, Masek MA, and Wilson LS, "Control of DNA Helix Openings during In-Vivo Neoplastic Cell Maturation", Proc. Am. Assoc. Cancer Res. 17: 15 (1976).

6. Fuchs RPP, "In-Vitro Recognition of Carcinogen-Induced Local Denaturation Sites in Native DNA by S₁ Endonuclease from Aspergillus Oryzae", Nature 257: 151-152 (1975).

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8. Tereba A, Skoog L, and Vogt PK, "RNA Tumor Virus-Specific Sequences in Nuclear DNA of Several Avian Species", Virology 65: 524-534 (1975).

Additional experimental details concerning ultrastructural probes of DNA conformation states (9, 10) and the role of of nRNA in regulating gene transcription (11) have been published previously:

9. Frenster JH, "Electron Microscopic Localization of Acridine Orange Binding to DNA within Human Leukemic Bone Marrow Cells", Cancer Res. 31: 1128-1131 (1971).

10. Frenster JH, "Ultrastructural Probes of Chromatin within Living Human Lymphocytes", Nature (New Biology) 236: 175-176 (1972).

11. Kanehisa T, Oki Y, and Ikuta K, "Partial Specificity of Low-Molecular-Weight RNA that Stimulates RNA in Various Tissues", Arch. Biochem. Biophys. 165: 146-152 (1974).

1. "A Steroid Receptor Coactivator, SRA, Functions as an RNA and is Present in an SRC-1 Complex".

1. "Selective Gene De-Repression by De-Repressor RNA".

2. Electron Microscopy of Human Lymphocytes before and after Activation by PHA (Busch H, 1974).

3. Anderson NG, and Coggin JH, eds., "Fourth Conference on Embryonic and Fetal Antigens in Cancer", Cancer Res. 36: part 2, pp. 3382-3540, (Sept. 1976).

4. Frenster JH, "Nuclear RNA Species Activate DNA Transcription within Chromatin", FASEB J. 13, A1506 (April 23, 1999)

5. Frenster JH, "Oncogenes as Molecular Targets within Active Chromatin", Clin. Cancer Res. vol. 5, suppl. 1, p. 3855a, (November, 1999).

6. Frenster JH, "Nuclear Ribosomes and RNA-RNA Duplexes".

7. Frenster JH, "The Educational Role of the Chromatin and Euchromatin Networks".

8. Frenster JH, "Activation of DNA Transcription within Repressed Chromatin by Nuclear RNA Species", RNA 2001: 237 (May 30, 2001).

9. Frenster JH "Activation of DNA Transcription within Repressed Chromatin", 14th John Innes Symposium, Sept. 5-8, 2001.

10. Frenster JH, "Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA", 2002.