Published by Invitation in: New Eng. J. Med. 288: 1224-1229 (June 7, 1973):

"Gene De-Repression."

John H. Frenster M.D. and Paul R. Herstein, B.S.
Division of Medical Oncology, Department of Medicine
Stanford University School of Medicine
Stanford, California 94305 


Introduction:
Mechanisms of Gene Control:
Detection of Gene De-Repression:
Occurrence of Gene De-Repression:
Mechanisms of Gene De-Repression:
Control of Gene De-Repression:
Clinical Aspects of Gene De-Repression:
Conclusions:
Support:
References:
Links: 

Cell diversity arises out of cell identity. The myriad of cell types composing an individual all result from the successive divisions of an original cell. A uniform content of DNA in each cell nevertheless results in a disparity of types of RNA, proteins, enzymes and antigens. These are some of the themes and paradoxes underlying current research in the control of gene function in mammals.

The issues may be sharply posed. DNA contains the biochemical information specifying the primary structure of such subsidiary molecules as RNA, proteins, enzymes and antigens, and ultimately of the cells and the organism itself. The adult mammal is derived from the fertilized single-celled ovum by repetitive cell divisions. But repetitive cell division alone would merely yield billions of identical copies of the original fertilized ovum, rather than the bewildering array of interacting cell types characteristic of the adult organism. How can the diversity of the adult be derived from the unity of the fertilized ovum, and how can such diversity occasionally be reversed during organ regeneration and neoplasia ?

Mechanisms of Gene Control

At least three mechanisms of gene control are potentially open to the fertilized ovum during its successive cell divisions. First of all, it could partition its genetic information unequally to each daughter cell, and by such successively unequal partitions gain a maximal degree of irreversibility in the resulting diversity of the daughter cells. Alternatively, it could partition its DNA equally to each daughter cell in each cell division, allowing the subsequent expression of such identical genetic information to be determined exclusively by factors local to particular microenvironments within the adult animal; this mechanism of local extracellular control would yield a maximal degree of reversibility in the resulting diversity of the daughter cells. Finally, it could combine both mechanisms to gain a mix of stability and reversibility of cell types that could be optimized for each species. Thus, amphibians that can regenerate an entire limb, and man, who cannot regenerate even a complete lobe of the liver, may represent the extremes of selections made concerning the optimal mix of stability and reversibility of cell types appropriate for the survival of each species.

The data of both classical biology and molecular biology indicate that it is indeed this third mechanism of gene control that is operative in vertebrates. The complement of DNA within each normal diploid cell of an individual is identical to that within every other normal diploid cell of that individual (1) and apparently to that within the original fertilized ovum (2). This fact probably excludes the first stategy of unequal partition of genetic information. At the same time, differentiated cells often maintain their differentiated state for many cell generations (3) when cultured in vitro under defined and simple conditions. These conditions do not include the native topography and cell associations that characterize local microenvironments within the organism, and this fact probably excludes the second mechanism of an exclusively extracellular control of an otherwise identical complement of DNA within each cell of an individual.

The third mechanism of gene control requires that specific molecules used in the control of DNA are themselves transmitted through cell division to the daughter cells, and suggests that such epigenetic molecules may not be partitioned equally to each daughter cell even when the DNA is partitioned equally. In addition, this mechanism of epigenetic control implies that under unusual circumstances, these epigenetic molecules may be influenced or even over-ridden by local factors of high intensity, permitting the conversion of cell types to an altered differentiated state by hormones, viruses or drugs.

These epigenetic mechanisms of gene control could theoretically be of at least two types. One type would function via a selective repression of an otherwise fully active genome. A second type would function via a selective de-repression of an otherwise fully repressed genome. Detailed molecular studies (4) have revealed that selective gene de-repression is the most important mechanism for gene control in vertebrates.

Glossary:
Genome  All the nuclear DNA within a cell.
Genetic Directly involving the nuclear 
DNA.
Epigenetic Involving non-DNA nuclear 
macromolecules.
Chromatin Complexes of nuclear DNA 
with non-DNA macromolecules
Oncogene DNA responsible for neoplastic 
behavoir.
Structural gene DNA coding for a cell protein.
Operator gene DNA directly responsive to 
control molecules.
Operon Operator gene + structural gene 
(or genes).
DNA template DNA molecule capable of 
synthesizing RNA.
Transcription RNA synthesis on a DNA molecule.
Reversed 
transcription		 DNA synthesis on a RNA molecule.
Repressor Nuclear macromolecule capable of 
inactivating a DNA template.
Repressed
gene		 DNA template + repressor.
De-repressor Nuclear macromolecule capable of
reversing the effects of a repressor.
De-repressed
gene		 DNA template + repressor +
de-repressor.
Transcription 
product RNA		 The immediate RNA product of
a DNA template.
Operator RNA RNA synthesized by an 
operator gene.
De-repressor 
RNA		 RNA capable of inducing 
gene de-repression.
Base sequence The linear order of purines and 
pyrimidines in RNA or DNA 
molecules.
RNA-RNA 
competitive 
hybridization		 A test for base sequences common 
to two or more RNA molecules.
RNA-RNA
duplex		 Double-stranded RNA, with each
strand complementary in base 
sequence to the opposite strand.
Homometric 
duplex RNA		 RNA-RNA duplex, with each 
strand of equal length.
Heterometric
duplex RNA		 RNA-RNA duplex, with strands
of unequal length.
Polyanions Macromolecules with high 
net negative charge.
Blastic
transformation.		 Lymphocyte after activation of
its nucleus and cytoplasm.

Detection of Gene De-Repression

Gene de-repression involves the formation of one or more species of RNA not present in the cell at an earlier observation time. When direct comparisons are made of the variety of RNA molecules being formed at each of the observation times, the most sensitive methods for detecting differences in these RNA molecules have been the technics of RNA-RNA competitive hybridization and RNA-directed DNA synthesis from defined single species of RNA (5).

Methods based on the detection of the synthesis of new proteins, enzymes, antigens or polypeptides not synthesized at an earlier observation time are less reliable since these may reflect changes in control of protein synthesis on polyribosomes rather than of RNA synthesis on de-repressed genes. Direct analysis of de-repressed gene sites through the use of ultrastructural molecular probes of high sensitivity and specificity (6) has also become an important auxilary technic in the epigenetic analysis of single living cells (Fig. 1). 



Figure 1. Ultrastructural Molecular Probe sites of Gene De-repression within a Promyelocyte from the Bone Marrow of an Untreated Patient with Chronic Myelocytic Leukemia (X 5,000). The electron-dense reaction product localized over DNA template sites that are free of histone repressors (6). Counts of the number of individual probe sites (small, black dots in the nucleus) per cell permit a quantitative estimate of gene de-repression for each cell within a cell population.

Occurrence of Gene De-Repression

During normal embryonic development, a progressive restriction occurs in the diversity of RNA molecules being synthesized per cell, from extensive RNA synthesis in the unfertilized ovum to a very marked restriction of RNA synthesis in the mature nucleated erythrocyte or to the complete absence of any nuclear RNA synthesis in the mature sperm (2). However, when such a nontranscribing mature sperm hybridizes with the mature ovum during fertilization, a very rapid de-repression of the sperm genome occurs, in what is probably the most crucial example of gene de-repression in mammalian systems. Such de-repression of the paternal genome converts the unfertilized ovum into a dividing diploid cell, and permits an equal maternal and paternal contribution to gene expression in all the resulting cells of the embryo and the adult.

During embryogenesis in the developing liver, a progressive restriction is observed in the diversity of RNA species being synthesized in the adult liver as compared to the embryonic liver, but when the adult liver is induced to regenerate after partial hepatectomy, de-repression of previously repressed genes again is manifested in the synthesis of RNA species characteristic of the embryonic state (7).

A similar progressive de-repression of previously repressed genes is also seen if neoplastic cells are assayed for the diversity of RNA species being synthesized as the neoplasm progresses first to a benign nodule, then to a spontaneous neoplasm and finally to a transplantable highly malignant neoplasm (8). Such de-repression of normally repressed RNA species also characterizes human leukemic lymphocytes (9), and may account for the reappearance within adult human neoplasms of cell surface antigens characteristic of normal fetal life as observed in adenocarcinoma of the colon, primary hepatoma of the liver, Hodgkin's lymphoma and chronic lymphocytic leukemia. A quite similar reappearance of normal fetal antigens has been observed in both chemical-induced and viral-induced neoplasms in laboratory animals (10). De-repression of normally repressed genes is the central feature of the oncogene hypothesis of neoplastic transformation (11), which postulates that either chemical or viral carcinogens can effect de-repression of normally repressed genes, perhaps by mimicking or bypassing the epigenetic molecules normally controlling such genes (12). In fact, the detailed molecular analysis of virally transformed neoplastic cells (13) has been of major importance in determining the mechanisms of gene de-repression within both neoplastic and normal cells.

Mechanisms of Gene De-Repression

A variety of physiologic and pathologic animal systems are now available for defining the molecular mechanisms of gene de-repression. Studies using these systems have taken advantage of the fact that repressed DNA molecules can be separated from de-repressed DNA molecules, and these can be separately analyzed within their native chromatin complexes by detailed biochemical, biophysical, metabolic and ultrastructural technics (4). Such separated chromatin complexes can also be used as assay systems for molecular species suspected of possessing repressor or de-repressor activity. These studies have uniformly revealed that histone proteins function as nonspecific repressors of DNA template activity, and that such histone repressors must be removed from the template before the underlying DNA can be active in directing RNA synthesis. 



Figure 2. Interaction of Histone Repressors with DNA Templates during Gene De-repression. Histone repressors (dark bars) form strong electrostatic bonds with the phosphate groups on the exterior of the DNA helix (3, 4). Experimentally, hydrolysis of such histone repressors by proteolytic enzymes such as trypsin, or physical displacement of such histone repressors by strong synthetic polyanions such as polyethylene sulfonate, results in de-repression of the underlying DNA template and increased rates of RNA synthesis. Under physiologic conditions, nuclear polyanions such as phosphoproteins, acidic proteins, lipoproteins, and nuclear RNA are capable of similarly displacing histone repressors and increasing the rates of RNA synthesis.

Under experimental conditions, histone repressors can be removed from DNA templates either by enzymatic digestion or be displacement with strong synthetic polyanions, whereas under physiologic conditions, such nuclear polyanions as phosphoproteins, acidic proteins, and nuclear RNA can function as de-repressors by effecting histone displacement, with subsequent de-repression of RNA synthesis (4).

Certain species of nuclear RNA apparently possess the ability to select specific portions of the genome for de-repression (14), perhaps by virtue of their ability to recognize specific base sequences on one strand of the DNA helix, and by hybridizing with such a single DNA strand effect a separation of the opposite DNA strand, 



Figure 3. Molecular Model of Selective Gene De-repression by De-repressor RNA (15). After histone displacement (dark bars), de-repressor RNA is postulated to hybridize to the nontranscribing strand of DNA, freeing the transcribing strand of DNA for messenger RNA synthesis (4, 15).

freeing it in turn as a template for messenger RNA synthesis at this restricted portion of the genome (15). The embryonic origin and control of such de-repressor RNA species are now under intensive investigation, and major attention is being paid to the possibility that oncogenic viral nucleic acids can effect gene de-repression and neoplastic transformation by virtue of their base-sequence resemblance to normal de-repressor RNA species found in embryonic life but not in adult life (12, 13).

Because the normal displacement of histone repressors from the underlying DNA templates during gene de-repression results in a profound conformational change within the chromatin complex (4), it has become possible to develop ultrastructural technics for mapping with high resolution the sites of gene de-repression within single cells. These methods use molecular probes that bind to the DNA template only if histone repressors have first been displaced from such templates (6). With such technics it is now possible to localize and quantitate gene de-repression within single living cells in human bone marrow, blood, and lymph node samples. During normal marrow granulocyte or erythrocyte differentiation, a progressive decrease occurs in the DNA template activity of the differentiating cells, whereas a progressive increase in such DNA template activity occurs in lymph-node lymphocytes reacting to invading neoplastic cells. An exact topographic mapping of the distribution of active and inactive cells within a tissue can be obtained, revealing the spatial patterns of gene de-repression present in large populations of cells in vivo. These studies have revealed an appreciable clustering of de-repressed cells within bone marrow and lymph nodes, which suggests some form of cell-cell interaction in the process of gene de-repression.

Control of Gene De-Repression

Data indicating a high degree of specificity and long-term stability of gene de-repression within adult cells (3, 16) have provided some insight into the nature of the feedback mechanisms that control gene de-repression. Cytogenetic analysis has revealed that all but one of the X chromosomes in adult mammalian cells are repressed (16) and that the selection of which X chromosome will remain de-repressed is made early in embryonic life and is then maintained through more than 50 generations of daughter cells. These feedback mechanisms appear exquisitely sensitive not only to the number of X chromosomes in the cell but apparently also to the gene content of X chromosome, strongly suggesting that such mechanisms reflect net messenger RNA accumulation in the cell while they are controlling further messenger RNA production via selective gene de-repression.

Recent studies on the molecular details of messenger RNA synthesis indicate how such feedback control may be exerted (Fig. 4). 



Figure 4. Postulated Feedback Control of Gene De-repression. A substantial portion of the high-molecular weight transcription product RNA is cleaved in forming the final messenger RNA (mRNA) molecule (5). The cleaved nonmessenger RNA fragment is thought to represent operator RNA (oRNA) synthesized on the operator gene (o) just before the synthesis of messenger RNA (mRNA) on the structural gene (sg) (10). De-repressor RNA (dRNA) specific for the particular operon (o + sg) is believed to bind to the nontranscribing strand of DNA within the operator gene (4, 15), and therefore to be complementary in base sequence with operator RNA. During excessive gene transcription, increased amounts of operator RNA (oRNA) within either the high-molecular-weight transcription product (0' + sg") or as free oRNA can form double-stranded heterometric or homometric RNA duplexes, by this means removing dRNA from the DNA template, and thus decreasing the rate of gene transcription at specific genes (10).

An important portion of the immediate transcription product RNA is excised to form the final messenger RNA molecule (5). The excised nonmessenger RNA fragment is thought to represent operator RNA synthesized on the operator gene of the operon under transcription (10). De-repressor RNA specific for the particular operon is believed to bind to the nontranscribing strand of DNA within the operator gene, and thus to be complementary in base sequence with operator RNA of the particular operon (15). Such complementarity allows the formation of stable RNA-RNA duplexes between the de-repressor RNA and the operator RNA when excessive production of messenger RNA by the operon has increased the concentration of the excised fragment of operator RNA (10). The formation of RNA-RNA duplexes under such conditions would remove de-repressor RNA from the operator gene on the DNA template, decreasing the rate of new RNA synthesis within the affected operon by eliminating the positive stimulus for such continued transcription. Several laboratories have recently reported the isolation of double-stranded RNA-RNA molecules involving the immediate transcription product RNA from a variety of tissues (17).

Clinical Aspects of Gene De-Repression

The ability of oncogenic viruses to de-repress fetal genes in the animal cell genome (11) may reflect the ability of the viral RNA (or DNA) to act as a fraudulent type of de-repressor RNA (10, 12). In such mimicry of  normal de-repressor RNA, these oncogenic nucleic acids are similarly associated by noncovalent bonds with the host genome, and on occasion can be removed from the host genome (13). Interestingly enough, the neoplastic state following after transformation is occasionally spontaneously reversible to normal within individual cells (18), and such reversion from the neoplastic state is now being intensively studied to ascertain whether it is mediated by the physical removal of oncogenic viral nucleic acid from the host genome (13).

Not only do neoplastic cells display the occurrence of gene de-repression clinically, but immune lymphocytes that are specifically sensitized against such neoplastic cells may also undergo a separate and perhaps more useful gene de-repression (19). The use of activated autologous lymphocytes for clinical immunotherapy of disseminated neoplasms requires blastic transformation of such lymphocytes via mechanisms that are thought to involve a characteristic gene de-repression sequence (19).

The recognition that steroid hormones penetrate into the cell nucleus, where they may effect gene de-repression in sensitive tissues (12), indicates to some degree the complexity of local factors modulating the basic epigenetic mechanisms controlling gene de-repression. Such local agents include the steroid hormones, chemical carcinogens, certain planar antibiotics and a variety of polyionic macromolecules (Table 1): 


 
Table 1. Correlation of Nuclear Ligand Binding and Subsequent RNA Synthesis.
Nuclear Ligand Preferred Form of DNA Effect on RNA Synthesis
Histones Double-stranded Decreased
Protamines Double-stranded Decreased
Actinomycin D Double-stranded Decreased
Acridine orange Double-stranded Decreased
Chloroquine Double-stranded Decreased
Polylysine Double-stranded Decreased
Testosterone Single-stranded Increased
Estradiol Single-stranded Increased
Methylcholanthrene Single-stranded Increased
RNA Polymerase Single-stranded Increased
De-Repressor RNA Single-stranded Increased
Polyoma DNA Single-stranded Increased

that bind preferentially to DNA in the single-stranded or double-stranded conformation, and by such preferential binding profoundly modify the rate activity of the DNA template for RNA synthesis. The net effect of these local agents on a cell is thought to reflect their relative binding affinities for the DNA template within the cell (12) and to provide a fine-tuning modulation of the basic de-repression mechanism.

In summary, in the future an understanding of gene de-repression and its control in the early developing embryo may well provide insight into the mechanisms whereby viral infections and chemical teratogens produce anomalies in the course of normal organ development. A further understanding of the control of gene de-repression during organ regeneration and hypertrophy may also permit consideration of the possibility of organ replacement via organ regeneration in the adult patient. In addition, the developing data concerning gene de-repression in neoplasia, immune-activation and hormone stimulation promise to provide new preventive and therapeutic approaches to human cancer, immune failure and endocrinopathies. As in so many other examples of the application of scientific advances to clinical medicine, these developments will almost certainly require corresponding changes in our clinical concepts and approaches to our individual patients.

Supported in part by research grants (CA-10174 and AM-01006) from the National Institutes of Health, and by a research-scholar award from the Leukemia Society to Dr. Frenster.

References

1. McCarthy BJ, Hoyer BH, "Identity of DNA and Diversity of Messenger RNA Molecules in Normal Mouse Tissues", Proc. Natl. Acad. Sci. U.S.A. 52: 915-922 (1964).

2. Davidson EH, "Gene Activity in Early Development", New York: Academic Press, 1968.

3. Davidson RG, Nitowsky HM, Childs B, "Demonstration of Two Populations of Cells in the Human Female Heterozygous for Glucose-6-Phosphate Dehydrogenase Variants", Proc. Natl. Acad. Sci. U.S.A. 50: 481-485 (1963).

4. Frenster JH, "Nuclear Polyanions as De-Repressors of Synthesis of Ribonucleic Acid", Nature 206: 680-683 (1965).

5. Melli M, Pemberton RE, "New Method of Studying the Precursor-Product Relationship between High Molecular Weight RNA and Messenger RNA", Nature (New Biology) 236: 172-174 (1972).

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

7. Church RB, McCarthy BJ, "Ribonucleic Acid Synthesis in Regenerating and Embryonic Liver. II. The Synthesis of RNA during Embryonic Liver Development and Its relationship to Regenerating Liver", J. Mol. Biol.23: 477-486 (1967).

8. Turkington RW, "Changes in Hybridizable Nuclear RNA during the Neoplastic Development of Mouse Mammary Cells", Cancer Res. 31: 427-432 (1971).

9. Neiman PE, Henry PH, "RNA-DNA Hybridization and Hybridization-Competition Studies of the Rapidly Labeled RNA from from Normal and Chronic Lymphocytic Leukemia Lymphocytes", Biochemistry 8: 275-282 (1969).

10. Herstein PR, Frenster JH, "Mated Models of Gene Regulation in Eukaryotes", in: Embryonic and Fetal Antigens in Cancer, Vol. 2, Edited by Anderson NG, Coggin JH, Springfield, Virginia: National Technical Information Service, 1972, pp. 5-7.

11. Todaro GJ, Huebner RJ, "The Viral Oncogene Hypothesis: New Evidence", Proc. Natl. Acad. Sci. U.S.A. 69: 1009-1015 (1972).

12. Frenster JH, "Correlation of the Binding to DNA Loops or to DNA Helices with the Effect on RNA Synthesis", Nature 208: 1093 (1965).

13. Nonoyama M, Pagano JS, "Separation of Epstein-Barr Virus DNA from Large Chromosomal DNA in Non-Virus-Producing Cells", Nature (New Biology) 238: 169-171 (1972).

14. Holmes DS, Mayfield JE, Sander G, Bonner J, "Chromosomal RNA: Its Properties", Science 177: 72-74 (1972).

15. Frenster JH, "A Model of Specific De-Repression Within Interphase Chromatin", Nature 206: 1269-1270 (1965).

16. Grumbach MM, Morishima A, Taylor JH, "Human Sex Chromosome Abnormalities in Relation to DNA Replication and Heterochromatinization", Proc. Natl. Acad. Sci. U.S.A. 49: 581-589 (1963).

17. Jelinek W, Darnell JE, "Double-Stranded Regions in Heterogenous Nuclear RNA from HeLa Cells", Proc. Natl. Acad. Sci. U.S.A. 69: 2537-2541 (1972).

18. Macpherson I, "Reversion in Hamster Cells Transformed by Rous Sarcoma Virus", Science 148: 1731-1733 (1965).

19. Frenster JH, Rogoway WM, "Immunotherapy of Human Neoplasms with Autologous Lymphocytes Activated In-Vitro", Proceedings of the Fifth Annual Leukocyte Culture Conference, Edited by: Harris J, New York: Academic Press, 1970, pp. 359-373. 

Additional References:

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

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euchromatin: "the most active portion of the genome within the cell nucleus".