John H. Frenster
Laboratory of Cell Biology
The Rockefeller University
New York, New York.

Dedicated to the Memory of Professor Emil Heitz (1892-1965).

The Partial Displacement of Histones by Nuclear Polyanions:
The Intermittent Localized Separation of the DNA Strands:
The Hybridization of De-Repressor RNA with a Complementary DNA Strand:
The Synthesis of Messenger RNA on a Single DNA Strand:

De-Repression by Strand Separation in Cell Biology:
Summary:
Acknowledgments:
Support:
References:
Additional References:
Other Links: 

The Genetic Equivalence of Diploid Cells:

The data of several types of investigation suggest that each diploid cell within a normal individual possesses
a full and identical complement of the total genetic information characterizing that individual (1). Thus, when normal differentiated tissues of a single species are compared, no significant differences are found in the chromosome numbers or specific karyotypes of each cell (11), in the quantity of DNA per diploid cell (12), in the average nucleotide composition, chromatographic behavior, or sedimentation pattern of the total DNA of each tissue (13), or in the complementary hybridization of the total DNA of each tissue (1). By contrast, differences between species are readily detected in each of these studies.

Recent experiments utilizing competitive hybridization of various RNAs to DNA disclose that within a single species, RNA of different tissues is relatively non-competitive, indicating that unique types of RNA are characteristic of each tissue (1). Such synthesis of tissue-specific RNA by cells all of which contain an identical complement of DNA suggests the presence of an epigenetic mechanism (2, 3) which selects the transcription of specific portions of the total DNA genome.

The Stability of the Selection Mechanism:

Several examples demonstrate the stability through cell division of the epigenetic mechanism which selects the transcription of particular portions of the genome. In female mammalian cells, the Barr sex chromatin body (14) represents one of the two X chromosomes (15). This chromosome is repressed and heterochromatinized early in the course of embryogenesis (16). Such repression affects either of the X chromosomes in a random fashion within each cell (17). Cloning studies have revealed that the repressed state persists throughout the life of the cell (18), and that the same X chromosome that is repressed in the parent cell is found repressed in all of the resulting daughter cells (18). Only in much older tissue cultures does the sex chromatin body disappear (19), with an apparent release of the repression of the X chromosome.

Again, if an active portion of an autosome is translocated to the repressed X chromosome (20), the translocated autosomal segment now displays a gradient of repression, most severe at the point of union with the repressed X chromosome, and declining with distance from the point of union (20). Such a gradient of repression is similarly stable through repeated cell divisions (20), and is a good example of the position effect in gene expression (21), which was first demonstrated in diptera (22).

Similarly, nuclear transplantation studies have revealed that during the course of embryogenesis in amphibians, certain changes occur in the nuclei which suggest the selective repression of a part of the genome (23, 24). Such repression is not uniform throughout all parts of the developing embryo, nor is it found equally in all amphibian species (24), but it is stable through many cell divisions either before or after nuclear transplantation (23-25).

Finally, epigenetic paramutational changes occur in maize which are consistent with a highly selective repression mechanism (26), and which are stable through many cell divisions of the somatic tissues and are often stable through meiotic divisions of the germ cells before being reversed or intensified (26).

By contrast, those mechanisms which nonspecifically affect the rate of synthesis of preselected RNA species do not demonstrate the same stability through the course of cell division as do those mechanisms which select the type of RNA species for synthesis. For example, the non-specific inhibition of all RNA synthesis by actinomycin D is reversible (8) and is not transmitted to the daughter cells. Similarly, the nonspecific stimulation of preselected RNA species by steroid hormones (6) or by polycyclic carcinogens (7) is reversible and is not transmitted through the course of cell division to the daughter cells.

Heterochromatin and Euchromatin:

During metaphase and anaphase of cell mitosis, the chromosomes are highly condensed, individually distinct, and relatively inert in RNA synthesis (27-30). By late telophase, the chromosomes have become less condensed, appear less distinct from each other, and begin the synthesis of RNA (27-30). By early interphase, all distinction between individual chromosomes is usually lost, and the chromosomal substance is visible either as condensed masses (chromocenters, heterochromatin) of repressed chromatin (31-33), or as extended microfibrils (euchromatin) of chromatin engaged in the active synthesis of RNA (32, 33).

This contrast between the extended and condensed physical states of specific chromosomal segments during cell interphase had been recognized in even the earliest microscopic observations of the chromosomes (34). Classical cytological studies revealed that the heterochromatic segments of particular chromosomes could be mapped by virtue of the prematurely condensed state (allocycly) of such segments during cell prophase (35), a time when the individual chromosomes are each fairly distinct (15). Utilizing such mapping of chromosomes during cell prophase, it was conclusively established that the heterochromatic sex chromatin body of Barr (14), found in female mammalian cells during cell interphase (14), was formed from one of the two X chromosomes in such cells (15). Subsequent genetic (17) and metabolic (36) studies revealed that the genes present on this heterochromatic X chromosome were repressed and were not expressed in the phenotype of the cell. Concurrently, it was found that this repressed and heterochromatic X chromosome displayed a characteristic delay in the onset of DNA replication during the S phase of its cell division (37-39). Later it was shown that those autosomal segments which also display such a delay in DNA replication similarly display a repression of RNA synthesis during cell interphase (36), and display the presence of secondary constrictions at these sites during cell metaphase (40). Such a selective repression of particular chromosomal segments during interphase provides a mechanism for effecting the functional mosaicism of phenotypes found in these cells of a single type of tissue (41).

Other animal systems demonstrate a similar correlation between active genetic transcription during interphase and an extended euchromatin state of the involved chromosomal segments, and conversely, between repression of genetic transcription during interphase and a condensed heterochromatin state of the involved chromosomal segments. Thus, in diptera the active gene loci on the polytene chromosomes appear as puffs or Balbiani rings of extended euchromatin microfibrils (42), while a regression of such puffs to the more usual condensed band structure parallels the subsidence of gene transcription at such loci to a repressed state (42, 43). Large repressed segments of such chromosomes often appear as confluent bands or blocks of heterochromatin (44). Similarly, during amphibian oogenesis the chromosomes assume an extended lampbrush state with a very active generalized synthesis of RNA (45). When such chromosomes are exposed to actinomycin D, the extended loops are retracted to form condensed chromomeres, and RNA synthesis is halted (46). Finally, in coccids the entire paternal haploid set of chromosomes becomes repressed and heterochromatic early in embryonic life (47), and can then either be discarded during spermiogenesis or can be de-repressed in partially haploid embryos (47).

Much of the DNA of interphase calf thymus lymphocytes is visible cytologically as condensed masses

Fig. 1. Intact calf thymus interphase lymphocytes before the addition of any preparative solutions (upper, A, B), isolated intact nuclei (middle, C, D), and extracted swollen nuclei after the removal of nuclear membranes and nuclear ribosomes (lower, E, F) all display both large mases of condensed heterochromatin and finer microfibrils of extended euchromatin (32).

of heterochromatin (31, 32, 48, 49). Metabolic studies reveal that up to 80% of the DNA of such nuclei is inactive in RNA synthesis (32, 50), such inactivity being due to a repression mechanism which is itself sensitive to trypsin digestion (51). It has recently become possible to isolate under gentle conditions (32) both the condensed heterochromatin and the extended euchromatin from these nuclei. 

Fig. 2. Isolated heterochromatin fraction (upper, A, B) is relatively free of contaminating euchromatin microfibrils, while isolated euchromatin fraction (middle, left, C) is free of contaminating heterochromatin masses. Particles in 10 mM calcium apparently are derived from fragmented nucleoli (middle, right, D) (32). Electron microscopic radioautograph (lower, E) of intact nucleus after incubation with glycerol-1,2-H³ (79) reveals the newly synthesized lipid is confined to tthe extended euchromatin microfibrils, with no synthesis seen over the condensed heterochromatin masses or over the nuclear membrane (79). X 6,000.

Under these isolation conditions, the morphology (32) and the differential metabolic activity (52) of the isolated heterochromatin masses and and of the isolated euchromatin microfibrils are unchanged when compared to that within the intact nucleus (32, 33, 49). When isolated nuclei are first incubated with radioactive RNA precursors and the resultant labeled nuclei are then examined either by chromatin fractionation (32) (Table 1):

^a Thirty-minute incubation, with materials and methods as previously described (32).

or by electron microscopic radioautography (49),

Fig. 3. Electron microscopic radioautograph of intact nuclei after incubation of nuclei with uridine-2-H³ (49) reveals that the newly synthesized RNA is confined to the extended euchromatin microfibrils, with little or no synthesis seen over the condensed heterochromatin masses (49). Marker equals 1.0 u. 

the newly synthesized RNA is found almost exclusively within the extended euchromatin microfibrils, with little or none found within the condensed heterochromatin masses.

Examination of the ultrastructure of these repressed heterochromatin masses and active euchromatin microfibrils within intact calf thymus lymphocytes (32, 33) (Fig 5, upper, A, B) reveals that euchromatin microfibrils are of 100 A. in diameter and of over 1.0 micron in length (33), and are ultrastructurally continuous with a dense reticulum of fibers composing the heterochromatin masses (32, 33). 

Fig. 5. Swollen extracted nuclei (left and right, A, B), reveal the extended euchromatin microfibrils to be of 100 A. diameter and up to 1.0 u in length (33), and to be ultrastructurally continuous with a dense reticulum of fibers within the condensed masses of repressed heterochromatin (33), X 11,000, X 30,000.

A similar type of ultrastructure has recently been demonstrated to link the active puffs and the repressed bands of polytene chromosomes in diptera (53). The DNA within repressed heterochromatin has most of its negative phosphate groups neutralized by polycationic histones (52), which allows such neutralized DNA to assume a highly condensed state. By contrast, theDNA within active euchromatin has much less of its negative phosphate groups neutralized, since within active chromatin many of the polycationic histones are partially displaced from DNA by the action of nuclear polyanions (52). Retaining a net negative charge along much of its length, the DNA within active chromatin assumes an extended state through the mutual repulsion of its many negative charges.

The DNA within Active and Repressed Chromatin:

Both repressed and active chromatin can now be isolated in quantity under gentle conditions from the calf thymus lymphocytes of a single animal (32). The development of this method of isolation has permitted extensive studies of the composition and the in-vitro metabolism of each of these forms of chromatin (52), with a view to delineating the mechanisms which mediate the repression or de-repression of nuclear RNA synthesis within these cells.

When the total DNA is isolated (54) from the repressed and from the active chromatin fractions of a single animal (32), no significant differences are found (52) in the average nucleotide base composition (55) of the DNAs from the two sources (Table 2).

^aMaterial and methods as previously described (52).

When the thermal hyperchromicity (56) of such isolated DNAs is studied, no significant differences in either the melting temperatures (T_m)or in the total hyperchromicity achieved are noted between DNAs isolated from either of the two types of chromatin (52). A similar constancy of average base composition and of thermal hyperchromicity has recently been found in the DNAs isolated from repressed and active portions of coccids (57).

By contrast, when the thermal hyperchromicity of the whole chromatin fraction is studied (56) without first isolating the DNA, several significant differences are now observed (52) between the DNA within either active or repressed chromatin. 

Fig. 6. Thermal hyperchromicity of DNA while within active and repressed chromatin and after its isolation. Materials and methods as described elsewhere (52). The DNA within active chromatin has a lower T_M and a lower total hyperchromic effect than the DNA within repressed chromatin or than the DNA of either type of chromatin after its isolation from chromatin.

First, the T_m of DNA while within active chromatin (81^oC) is significantly less than that of DNA while within repressed chromatin (86^oC), although both are higher than that of isolated DNA from either source. Second, the total hyperchromicity achieved by the DNA while within active chromatin is much less than that achieved by the DNA while within repressed chromatin, although when this same DNA is first isolated from active chromatin, it now displays as much total hyperchromicity as does the DNA within repressed chromatin or the DNA isolated from repressed chromatin. The T_m data reveal that the DNA while within active euchromatin is less well stabilized to thermal denaturation than is the DNA while within repressed chromatin, although both are more stable than is the DNA isolated from either type of chromatin (52). The total hyperchromicity data indicate that the DNA while within active chromatin is already in a partially denatured or single-stranded state, and that as the histones and polyanions of active chromatin are removed during DNA isolation, this partially single-stranded DNA reverts to that double-stranded state seen in isolated DNA or in DNA while within repressed chromatin.

These interpretations of the data are consistent with other studies utilizing the reactivity of DNA with human anti-DNA antibodies (58) in an agar immunodiffusion system (58). In such studies, the DNA while within repressed chromatin is found to be nonreactive with such anti-DNA antibodies, while both the DNA while within active chromatin and the DNAs isolated from either repressed or active chromatin are fully reactive (52). Polycationic histones are known to form electrostatic complexes with DNA which result in increased stability of the DNA to thermal denaturation (59, 60), and in reduced reactivity of the DNA to anti-DNA antibodies (58). Since both repressed and active chromatin contain nearly equal amounts of histones in relation to their DNA contents (52), these data also indicate that the DNA while within active chromatin is less firmly complexed by its histones than is the DNA while within repressed chromatin.

The Histones within Active and Repressed Chromatin:

Histone proteins extracted from lymphocyte nuclei are capable of repressing the template function of DNA in a variety of cell-free systems (61-63). Such repression is mediated by the ability of histones, as cationic polyelectrolytes, to form electrostatic complexes with DNA (64-66), and by this means preventing the separation of the strands of the DNA double helix (59, 60) during transcription in RNA synthesis. Histones are sensitive to trypsin digestion (65), as is the repression mechanism found within intact lymphocyte nuclei (51). These data suggest that histones may function as repressors of DNA template function within the cells of higher organisms (51, 59, 61-63), but histones have not as yet been demonstrated within bacterial cells (67, 68).

When both the total histones and the lysine-rich histones are extracted (69) from active and repressed chromatin fractions isolated from the same animal, the total histone contents of the two chromatin fractions relative to the DNA contents are found (52) to be not significantly different (Table 3), 

^a Materials and methods as previously described (52).
^b Mean +/- S.E.

with 20% of the total histones within each chromatin fraction being of the lysine-rich variety (52). Electrophoresis of the total histones on cellulose polyacetate (M. L. Green, personal communication) reveals no significant differences in the banding patterns between the histones extracted from the two types of chromatin. 

Such constancy of both the types and the quantities of histones found within repressed and active chromatin is similar to the constancy of the types and the quantities of histones found previously within animal cells varying widely in their tissue of origin (65), age (70), rate of RNA synthesis (70), or neoplastic character (71, 72). These data indicate that histones are a necessary but not a sufficient mechanism (73) for mediating the differential rates of RNA synthesis observed within active and repressed chromatin (32, 49), or for mediating the differential stability to thermal denaturation (52) or the differential reactivity with anti-DNA antibodies (52) of the DNA within active and repressed chromatin.

Nuclear Polyanions and Hormones within Chromatin:

By contrast, when the nuclear polyanion contents (52) of active and repressed chromatin fractions prepared from the same animal are determined relative to the DNA contents, active chromatin is found to contain a two-fold excess of total nonhistone proteins remaining after extraction of histones (74-77), a five-fold excess of total RNA and of total phospholipids (78), and an almost four-fold excess of total phosphoprotein phosphorous (79). Such nuclear phosphoproteins (79-81) constitute up to 15% of the nonhistone residual proteins within active chromatin (79).

Previous studies have revealed increased amounts of the nonhistone residual proteins within the nuclei of tissues engaged in active RNA synthesis (74-77), and in the nuclei of certain neoplasms (82). A parallel increase in both the nonhistone residual proteins and in total RNA has recently been found in the soluble chromatin of active tissues of the chicken (70), in the active puffs of polytene chromosomes (42), and in the active loops lampbrush chromosomes (45). The presence of these nuclear polyanions in excess within active chromatin suggested that they may be playing a role in antagonizing the electrostatic interaction between DNA and polycationic histone repressors within active chromatin (52).

In addition to these nuclear polyanions, active chromatin is found to contain an excess of lipids (78), including phospholipids (52), neutral fats, and cholesterol. Incubation of intact nuclei, followed by chromatin fractionation or by electron microscopic radioautography (78) reveals that lipid synthesis is confined to active chromatin.

Recent studies of the preen gland of ducks disclose that when testosterone is used to stimulate RNA synthesis, both labeled testosterone and newly synthesized RNA are localized within the active chromatin fraction (83). The mechanism of such steroid hormone localization and action may be related to the known increased affinity of both steroid hormones and polycyclic carcinogens for hydriphobic binding to single-stranded DNA (84). Some of the DNA within active chromatin appears to be in the single-stranded state (73), but the DNA within repressed chromatin appears to be double-stranded (73).

The Stability of Repression and De-repression:

Incubation of intact nuclei with radioactive precursors of RNA, followed by isolation of the the two forms of chromatin from such labeled nuclei, reveals that most if not all RNA synthesis occurs within the extended euchromatin microfibrils (32, 49). The condensed heterochromatin masses display a repression of such RNA synthesis while within the intact nucleus (32, 49). When, by contrast, such intact nuclei are first fractionated to yield the two forms of chromatin, and each isolated chromatin fraction is then tested for its ability to synthesize RNA, it is found (Fig. 7), 

Fig. 7. Incorporation of ATP-8-C¹⁴ into RNA of of isolated chromatin fractions. Materials and methods as described elsewhere (52). The chromatin isolation procedure preserves the repressed and active states of each type of chromatin as found within the intact nucleus.

that the extended euchromatin fraction remains three to eight times more active in RNA synthesis than the condensed heterochromatin fraction of the same animal (52). These data indicate that the chromatin fractionation procedure (32) is gentle enough so that the mechanisms controlling RNA synthesis within the intact nucleus have survived the isolation procedure, and are still functioning within these isolated chromatin fractions (52).

RNA synthesis within such isolated chromatin fractions is characterized as follows (52):
(a) The radioactive product of incorporation is insoluble in cold 5% trichloroacetic acid, and 70 to 90% of the incorporated radioactivity is removed on subsequent digestion with ribonuclease (Table 4):

^a Materials and methods as previously described (52).
^b ATP-8-C¹⁴ incorporated into RNA of isolated chromatin during 30-min. incubation - see methods (52).
^c Incorporated counts removed by digestion with RNase for an additional 30 min - see methods (52).
^d Mean +/- S.E.

(b) the incorporation process requires the simultaneous presence of all four ribonucleoside triphosphates, and is sensitive to the presence of actinomycin D or deoxyribonuclease, and is completely resistant to the presence of puromycin (Table 4);
(c) the RNA polymerase and the DNA templates needed for RNA synthesis are contained within the isolated chromatin fractions in a native state (52).
In these incorporation properties, the RNA synthetic process is similar to that demonstrated earlier within other chromatin preparations isolated from animal tissues (70, 85).

The relative resistance of the incorporation process to the presence of ribonuclease (Table 4) indicates that most of the newly synthesized RNA remains in hybrid formation with template DNA during the course of the 30-min incubation. Natural RNA-DNA hybrids are known to be relatively resistant to the action of ribonuclease (86). It is only after the incubated chromatin fractions are precipitated in cold 5% trichloroacetic acid that the newly synthesized RNA becomes sensitive to ribonuclease digestion (52). In addition, the incorporation process within active chromatin is much more resistant to the presence of acridine orange (Table 4) than is the small amount of incorporation within repressed heterochromatin, providing additional evidence that RNA synthesis within active euchromatin is occurring on single-stranded DNA (73). Acridine orange and other acridine inhibitors are known to intercalate between the bases of the DNA double helix (87,88), and to bind with greater affinity to double-stranded DNA than to single-stranded DNA (88, 89). The consequences of such acridine orange binding are a non-specific inhibition of the template functions of DNA (90), probably by inhibiting the separation into single strands of the DNA double helix. When exposed to equal concentrations of acridine orange, then, single-stranded DNA binds much less of the inhibitor than does double-stranded DNA (88, 89), and is more resistant to the inhibitory effects on template function as a consequence (73).

Experimental Repression and De-Repression:

The stability of the mechanisms mediating the control of RNA synthesis through the course of the chromatin isolation procedure (52) provides a system which is useful in studying the experimental variation of repression and de-repression within chromatin fractions isolated from the thymus lymphocytes of a single animal (52).

The addition of trypsin to incubations of intact nuclei has been shown to result in a selective removal of repressor histones by the preferential hydrolysis of peptide bonds involving lysine and arginine, with a consequent marked increase in the rate of nuclear RNA synthesis (51). Similar additions of trypsin to incubations of isolated chromatin fractions result (52) in a marked increase in RNA synthesis within both isolated active and repressed chromatin (Table 4), indicating that active chromatin is partially repressed by its constituant histones, and that both forms of chromatin can increase their rates of RNA synthesis following removal of their histones (52). When the synthetic polyanion polyethylene sulfonate is added to incubations of isolated active and repressed chromatin fractions, a marked increase in RNA synthesis occurs within each chromatin fraction (52), rivaling in magnitude the increase produced by the removal of histones during trypsin digestion (Table 4). Polyethylene sulfonate is a polyanion of high molecular weight and charge density (9), and lacks any of the modifying basic or hydrophobic side groups, or both, that characterize the naturally occurring nuclear polyanions found in excess within active chromatin (Table 3).

Because of this de-repressor activity manifested by a synthetic polyanion, it was of interest to test the various natural nuclear polyanions (Table 3) for a similar ability to de-repress the synthesis of RNA within isolated chromatin fractions (52). The total RNA of both the nuclear and the cytoplasmic fractions of the thymus lymphocytes of a single animal was isolated in parallel by a phenol exraction procedure employing an aqueous phase consisting of 0.1 M Tris HCl, pH 8.5 (91), and collecting only that RNA which is precipitated from complete solution by 10% NaCl and which is free of contaminating DNA (91, 92). A comparable preparation of yeast RNA was obtained commercially (92). The addition of thymus total nuclear RNA to incubations of active and repressed chromatin fractions isolated from a single animal results in an increase in the RNA synthetic activity within isolated repressed chromatin, but has little effect on active chromatin (52), while the addition of yeast RNA actually decreases the RNA synthetic activity within isolated active chromatin, perhaps by competing with the more effective endogenous thymus nuclear RNA already contained within active chromatin.

When each of the major classes of nuclear polyanions which are found in excess within active chromatin (Table 3) is compared directly by addition to incubations of active and repressed chromatin isolated from a single animal (Table 5):

^a Materials and methods as described previously (52).
^b Mean +/- SE. 

These data reveal that each of the nuclear polyanions found in excess within active chromatin (Table 3) is capable of de-repressing RNA synthesis when added to incubations of repressed chromatin. The resistance of active chromatin to such additions implies that the de-repression mechanism within active chromatin is already fully saturated by natural nuclear polyanions, and can only respond (Fig. 8) to excessively strong synthetic polyanions such as polyethylene sulfonate. 

The De-Repressor Role of RNA:

When high molecular weight RNAs are compared directly by addition to incubations of isolated repressed chromatin (Fig. 9),

Fig. 9. Effect of various RNAs on incorporation of UTP-2-¹⁴C into RNA of isolated repressed chromatin. Materials and methods as described elsewhere (52). Thymus total nuclear RNA is much more effective as a de-repressor than is either the total cytoplasmic RNA or the nuclear ribosomal RNA of the thymus of the same individual. 

By contrast, each tissue within an organism appears to possess several species of RNA that are unique to that tissue (1). RNA is capable of either hybridizing in perfect register with highly selective species of complementary DNA in a stable union that is resistant to the action of ribonuclease (86), or of aggregating in looped imperfect register with a broader spectrum of DNA species in a labile union that is less resistant to ribonuclease (106). Some species of nuclear RNA appear to be particularly effective (52) in de-repressing the synthesis of RNA within repressed chromatin (Fig. 9), and tissue-specific species of RNA have been found to be capable of the embryonic induction of specific organ systems (107, 108). In addition, during cell division, much of the nuclear RNA of the maternal cell is transmitted intact to the daughter cells (109-111), providing a mechanism for that continuity of specific de-repression in the daughter cells which is implicit in cell differentiation and which is stable through the course of cell division (112, 113).

These data reveal that particular species of RNA possess a high degree of informational specificity (114), a diversity unique for each tissue of an organism (1), a highly selective mechanism for firmly hybridizing with specific portions of the DNA genome (86), a demonstrated ability to de-repress the synthesis of RNA within repressed chromatin (52), a specific capacity to induce the embryonic formation of particular organ systems (107), and a known stability through the course of cell division (109-111). Because of these germane characteristics, a species of de-repressor RNA strongly qualifies as the selective agent in the transcription of specific portions of the DNA genome (52, 73).

The Strand-Separation Model of De-Repression:

These experimental data imply a mechanism (73) of of specific de-repression of RNA synthesis within interphase chromatin (Fig. 10, right) with the following essential features:

(a) Polycationic histone repressors, which inhibit DNA strand separation, are partially displaced by nuclear polyanions from portions of the DNA genome, but these displaced histones are losely retained within the chromatin complex:

(b) those portions of the DNA genome that are thus freed of histones experience an intermittant localized separation of the complementary DNA strands, while the ends of the double helix remain in exact register;

(c) during such intermittant DNA strand separations, de-repressor RNA can hybridize with complementary sequences on one of the separated DNA strands, tending by such hybridization to stabilize or even to extend the loop of DNA strand separation; and

(d) the remaining complementary DNA strand of the separation loop is now free to serve as a template for the continuous synthesis of specific messenger RNA (73).

Relevant Evidence for Features of the Model:

The experimental evidence relevant to each of the four essential features of the strand separation model of specific de-repression may now be summarized:

The Partial Displacement of Histones by Nuclear Polyanions:

Histones interact with DNA by forming electrostatic complexes through the negative phosphate groups of DNA (66). This complex formation antagonizes the separation of of the DNA strands of the double helix during thermal denaturation (59, 50) and transcription to RNA (73). Within repressed chromatin, DNA has most of its negative phosphate groups neutralized by histones (52), allowing this DNA to collapse (64) into condensed masses of heterochromatin. Within active chromatin, the interaction of histones with nuclear polyanions leaves some phosphate groups along the length of the DNA molecule in a charged state, inducing an extended form of euchromatin (64) in such DNA by the mutual repulsion among the multiple negative charges. Both active euchromatin and repressed heterochromatin contain equal amounts of histones relative to their DNA contents (52), but the histones within active euchromatin are less effective in inhibiting the separation of the DNA strands during the repression of RNA synthesis (52), during the thermal denaturation of DNA (52), and during the reaction of the DNA with anti-DNA antibodies (52, 58). By contrast, such nuclear polyanions as RNA and phosphoproteins are found in excess within active chromatin (52), and when added to repressed chromatin are capable of de-repressing the synthesis of RNA (52).

The Intermittant Localized Separation of the DNA Strands:

Recent studies of H³ exchange within the DNA double helix reveal that the helix is in a constant process of opening and closing, or "breathing" (115, 116). Polycationic histones and other multivalent cations inhibit this intermittant DNA strand separation by simultaneously combining with phosphate groups of both strands (59, 60, 116). Other inhibitors such as actinomycin D (117, 118) and acridine orange (119) inhibit this intermittant DNA strand separation by simulataneously combining with the nucleotide bases of both strands. Thermal hyperchromicity studies reveal that much of the DNA within active chromatin is in the single-stranded state (52, 73), and the resistance of active chromatin to the inhibitory effects of acridine orange, an inhibitor which intercalates preferentially between the bases of double-stranded DNA (87, 88), also supports this interpretation. When the DNA is isolated from active chromatin, it now displays a full degree of hyperchromicity characteristic of double-stranded DNA (52, 73), indicating that some of the DNA within active chromatin remained in perfect register in the double-stranded State (52, 73). When the histones and polyanions of active chromatin are removed during DNA isolation (54), the presence of some portion of each DNA molecule in perfect register facilitates the renaturation of the separated strands (86).

The Hybridization of De-Repressor RNA with a Complementary DNA Strand:

It has recently been shown that a species of nuclear RNA is particularly effective as a de-repressor (52), probably by virtue of its particular nucleotide base sequence. The resistance of active chromatin to further de-repression by added RNA (52) implies that the finite number of sites on the DNA genome complementary to the added RNA are already in a hybrid form with RNA, while the attainment of a similar level of synthesis within repressed chromatin following the adddition of de-repressor RNA (52) suggests a similar saturation of complementary DNA sites. De-repressor RNA can interact with repressed chromatin in at least three distinct ways. It can simply displace histones from portions of the DNA genome at random by virtue of its polyanionic character (52). Some of its nucleotide bases can pair with localized groups of complementary bases on a single strand of DNA in a loose unstable aggregate in which much of the RNA remains sensitive to the action of ribonuclease (106). Or more and more neighboring bases on the RNA can pair with complementary bases on the single strand so that a significant length of the DNA genome is in perfect register with de-repressor RNA, and the resultant RNA-DNA hybrid is relatively resistant to the actions of ribonuclese (86). The formation of such a perfect register RNA-DNA hybrid occurs only with single-stranded DNA (1). The stability of such RNA-DNA hybrids throughout the cell division is unknown. During the S phase of DNA replication, de-repressor RNA and messenger RNA could both be released from the genome to return in the daughter cells, since nuclear RNA is known to be transmitted intact from the parent cell to the daughter cells (109-111). Such stability of nuclear RNA provides a mechanism for the stability of selective transcription into the daughter cells (17-26).

The Synthesis of Messenger RNA on a Single DNA Strand:

It has recently been shown that only one strand of the DNA double helix is transcribed during RNA synthesis (120-122). The remaining complementary DNA strand has no known function during RNA synthesis (106). If this complementary DNA strand does hybridize specifically with de-repressor RNA, certain segments of the de-repressor RNA molecule specific for a particular gene locus must be complementary to those portions of those messenger RNA molecules synthesized at that locus, since portions of each RNA molecule would be complementary to single DNA strands, and these DNA strands would be complementary to each other. In this regard, it is interesting that a species of nuclear RNA has recently been isolated from rat liver which is characterized by a base composition similar to that of DNA and of messenger RNA, but which possesses sedimentation and metabolic properties which distinguish it from messenger RNA (123). If messenger RNA and de-repressor RNA are complementary over certain portions of their length, they might be capable of forming hybrids of double-stranded RNA which would be more stable through the mitotic cycle (124). Steroid hormones and polycyclic carcinogens are known to bind preferentially to single-stranded DNA (84), and they may accelerate the rate of RNA synthesis of preselected species of RNA by promoting the dissociation of the template DNA-messenger RNA hybrid (84).

De-Repression by Strand Separation in Cell Biology:

Many areas within cell biology are providing fundamental data regarding the mechanisms effecting selective transcription of specific portions of the DNA genome. The strand separation mechanism of specific de-repression is germane to certain fundamental questions in some of these areas.

In embryogenesis, a fundamental question is the developmental origin of the unique specificity of transcription characterizing each differentiated tissue (1). A tissue-specific distribution of unique de-repressors RNAs might arise by the successively unequal distribution at the time of each early cell division (125) of what was originally a full complement of all possible de-repressor RNA species, synthesized during oogenesis (45, 46, 104).

Similarly, in the differentiation of stem cells into mature end stage cells, a fundamental question is the mechanism whereby progressively fewer species of messenger RNA are synthesized as the cells mature (126). Such diminution might occur as a result of the progressive loss of species of de-repressor RNAs during successive cell divisions, either by dilution or by hydrolysis of such de-repressor RNA (125).

In oncogenesis, a fundamental question is the mechanism whereby a new range of messenger RNA species is synthesized in the neoplastic cells compared to normal cells of the same tissue (127). If neoplastic change does precede karyotypic change (128, 129), an increase in the range of de-repression could occur if such oncogenic agents as viral nucleic acids, chemical carcinogens, or radiation acted to either displace histones, promote DNA strand separation, or mimic the nucleotide base sequences of certain normal species of de-repressor RNA (130).

In immunogenesis, a fundamental question is how the RNA (131, 132) of cells which have phagocytized the sensitizing antigen (133) is capable of inducing a state of immunological memory (132) in those plasma cells contiguous to the phagocyte (134). Some of the immunogenic RNA transferred to the plasma cells could be retained in the immune clone by hybrid formation with single strands of plasma cell DNA, functioning there as a stable de-repressor through successive cell divisions.

Finally, in the action of steroid hormones and polycyclic hydrocarbons, a fundamental question is how these hydrophobic agents are able to accelerate the synthesis of preselected species of RNA (6, 7). Both steroid hormones and polycyclic hydrocarbons are known to bind preferentially to single-stranded DNA rather than to double-stranded DNA (84), and are thought to be capable of shifting the equilibrium in favor of single stranded DNA by such binding (84). They could have a similar effect on the stability of template DNA-messenger RNA hybrids, freeing the DNA template for accelerated RNA synthesis.

Summary:

Although each diploid cell within an individual probably contains a full and identical complement of all the DNA species characterizing that individual, not all of these DNA species are transcribed to RNA within any one cell or tissue. Stable epigenetic mechanisms select specific portions of the DNA genome for transcription. Such selective mechanisms are seen to employ a nonspecific generalized repression of all DNA within a cell by means of complex formations with polycationic histone repressors. Nuclear polyanionic de-repressors antagonize the interactions between DNA and histones, and effect a partial displacement of repressor histones from localized portions of the DNA genome. Such histone displacement allows these DNA segments to undergo an intermittant separation of the strands of the DNA double helix. A species of nuclear RNA appears to function as a highly selective agent in de-repression by hybridizing with a single DNA strand, freeing the complementary DNA strand for continuous specific messenger RNA synthesis.

Acknowledgments:

It is a pleasure to acknowledge the helpful discussions and gracious cooperation of my colleagues: Drs. A. E. Mirsky, V. G. Allfrey, V. C. Littau, H. G. Rose, M. L. Green, T. A. Langan, E. M. Tan, and R. Faulkner, and of Drs. V. R. Potter, W. Szybalski, H. Swift, and H. P. Rusch.

Support:

This investigation was supported in part by Research Career Development Award CA-17857 from the United States Public Health Service.

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