John H. Frenster
Laboratory of Cell Biology
Rockefeller Institute
New York, NY 10021 

During cell interphase in higher organisms, the DNA molecules of the cell nucleus are found arrayed either within condensed masses of heterochromatin (7, 8) in which RNA synthesis is repressed (7-9), or within extended microfibrils of euchromatin (7, 8) in which RNA synthesis is active (7-9). When such repressed heterochromatin and active euchromatin complexes of a single tissue are each isolated in parallel under identical gentle conditions (7), the isolated heterochromatin retains its original repressed and condensed state (4), while the isolated euchromatin retains its original active and extended state (4). The native associations between DNA and the other molecular species of chromatin, such as repressor histones, de-repressor RNA, and endogenous RNA polymerase, have been examined within isolated heterochromatin and isolated euchromatin in a variety of physical, chemical, and metabolic investigations (4, 6).

DNA molecules isolated from either type of chromatin of one tissue are not significantly different in their base composition (4, 6) or in their thermal denaturation profiles (4). Similarly, both types of chromatin contain the same quantities and patterns of histones in relation to their DNA contents (4, 6). By contrast, active euchromatin contains an excess of such nuclear polyanions as RNA (4), phosphoproteins (10), and phospholipids (11), and localizes testosterone during stimulation of RNA synthesis by this hormone (12).

When DNA molecules are examined while within the active euchromatin complex, they display a 60 percent reduction of their usual thermal hyperchromicity (4), indicating a significant degree of separation of the strands within their DNA double helices (5, 6). By contrast, such DNA molecules of the sample display an entirely normal degree of thermal hyperchromicity when they are first isolated away from the histones and polyanions of the euchromatin complex before being examined (4). Such contrasting behaviour suggests that the DNA strand separations observed within the intact euchromatin complex depend on an association with the other molecular species of euchromatin for the stabilization of such strand separations (5, 6). The regaining of normal hyperchromicity following DNA isolation also suggests that these strand separations are only partial or looped within any given DNA molecule, with some portion of each helix remaining in exact register during selective gene transcription (5, 6). In parallel investigations under identical conditions, the DNA molecules while within the repressed heterochromatin complex do not reveal any reduction of their usual hyperchromicity (4), but display an increased stability to strand separation while within the heterochromatin complex (4).

Both active euchromatin and repressed heterochromatin contain equal amounts and types of polycationic histones in relation to their polyanionic DNA (4, 6). Such histones function as non-specific repressors of the template function of DNA by interacting electrostatically with the negative phosphate groups on the exterior of the DNA double helix (13, 14). This histone-DNA interaction neutralizes the multiple negative charges along the length of the DNA molecule (13, 14), overcoming the mutual repulsion otherwise present between these highly charged DNA molecules (15), and favouring the formation of condensed masses of repressed heterochromatin (6, 16). In such an interaction, the histones cross-link two or more DNA strands (17), thus stabilizing the double-stranded DNA helices against strand separation and consequent transcription (18).

The polycationic histone repressors can be partially displaced from the DNA double helices by several types of those polyanions found in excess within active chromatin (4). In such action, these polyanions function as natural de-repressors, permitting the now-charged DNA double helix to assume the extended state (6) and to undergo its normal degree of spontaneous strand separations (19). Strand separation loops formed spontaneously appear to be stabilized and perhaps extended within specific loci of the genome by the co-operative binding of specific de-repressor RNA molecules to one of the two separated DNA strands at these loci (5, 6). The remaining complementary DNA strand within the separation loop is then free to serve as a template for selective RNA synthesis (5, 6).

These investigations indicate that the physical state of the DNA molecules during selective transcription in higher organisms is characterized by local stabilized separations of the strands of the DNA double helix within specific portions of the genome (5). By contrast, repressed portions of the genome are characterized by an increased stability of the DNA helix (4), with no evidence of strand separations(5) or of active transcription (7, 9), and appear as condensed masses of heterochromatin during cell interphase (7, 8). Later in the cell cycle, during DNA replication, these same repressed heterochromatin portions of the genome display a late phase of DNA synthesis compared to the rest of the genome (20). Since in vivo DNA replication involves some degree of strand separation of the replicating DNA molecules (21), the delay in DNA replication observed within these repressed heterochromatin regions may reflect the increased stability of the DNA helices within these regions found earlier in the cell cycle during selective transcription (4).

Still later in the cell cycle, after the completion of DNA replication, these same repressed heterochromatin regions of the genome (22) may now extend and form secondary constrictions (23) in localized areas of the metaphase chromosomes (22). Such secondary constrictions map identically within each of the sister chromatids (23), indicating that each daughter cell receives an identical complement of chromosomes the DNA of which is physically altered in those same gene loci that were repressed in transcription and delayed in replication in the maternal cell (6). Indeed, following cell division, it has been shown that at least some of the same gene loci that were repressed for transcription in the maternal cell are similarly repressed in each of the daughter cells for many subsequent cell generations (24), indicating a continuity of the specific transcription selection through the entire course of many cell divisions (6). Such a continuity could be mediated by the passive symmetric transfer of maternal de-repressor RNA species to each of the daughter cells during cell division (6, 25).

The strand separation loops within the DNA molecules of active euchromatin are the likely sites of action of those molecular species which increase or decrease the rate of RNA synthesis non-specifically (6). Thus, such steroid hormones as oestradiol and testosterone bind preferentially to the single-stranded form of DNA (26), tending to stabilize the strand separation loops within DNA (26), and non-specifically increasing the rate of RNA synthesis within pre-selected loci of the genome (27). In this regard, testosterone is found localized preferentially within active euchromatin during the stimulation of nuclear RNA synthesis by hormone (12). Conversely, such inhibitors as histones (14), actinomycin D (28), acridine orange (29), and chloroquine (30), all bind preferentially to the double-stranded form of DNA, tending to stabilize the DNA helix against strand separations, and non-specifically decreasing the rate of synthesis of all RNA species (6).

Recent investigations suggest that the strand separation loops within active euchromatin may be sensitive sites of action during radiosensitization (31) and during the interaction of the host genome with oncogenic viruses (32).

This work was carried out during the tenure of a research career development award (CA 17857) from the U.S. Public Health Service.

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1. Frenster JH, "Control of DNA Strand Separation during Repression and Derepression of RNA Synthesis", J. Cell Biol. 27: 30A (1965).