and cells undergoing such gene de-repression often display a reversible transformation of heterochromatin to euchromatin within their nuclei (Figs. 1 and 2).
John H. Frenster
Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, California.
I. History of Heterochromatin and Euchromatin:
II. Ultrastucture of Heterochromatin and
Euchromatin:
I. History of Heterochromatin and Euchromatin:
Recent progress in the study of the cell nucleus has increased the long-standing interest in heterochromatin and euchromatin. Heitz (1929) originally described as heterochromatin that portion of the nuclear chromatin which demonstrated its allocycly by maintaining a condensed state throughout cell interphase while the remainder of the nuclear chromatin was extending to what he termed the euchromatin state. Cooper (1959) was able to summarize the data from Drosophila which suggested that heterochromatin and euchromatin differed in their biophysical conformations and in metabolic expression of their genes but not in their basic structure of DNA arranged within chromosomes.
Since that time, increasingly detailed genetic studies of the heterochromatic X chromosomes of female mammalian cells (Lyon, 1961: Grumbach et al, 1963; Brown and Chandra, 1973), of the heterochromatic paternal chromosomes of coccids (Brown and Nur, 1964), and of the heterochromatic regions of the Y chromosome in Drosophila (Hess and Meyer, 1963) have revealed that the genes within heterochromatin are repressed but can later be expressed when the heterochromatic region undergoes a transition to euchromatin. Similarly, cytological studies have revealed that heterochromatin displays late replication of its DNA (Lima-De-Faria, 1969); displays no uptake of the DNA ligand, acridine orange (Frenster, 1971); and displays little or no synthesis of RNA (Hsu, 1962; Littau et al, 1964; Klevecz and Hsu, 1964), all of which are reversed when heterochromatin is converted to euchromatin. Finally, isolation of heterochromatin and chromatin from mammalian cells (Frenster et al, 1963; McConaughy and McCarthy, 1972) has permitted a direct biochemical and biophysical analysis of DNA template activity in RNA synthesis either before or after such isolation (Frenster, 1965b), again demonstrating that template restriction reflects repression of DNA molecules within heterochromatin (Frenster, 1971), which is reversed when heterochromatin is converted to euchromatin (Frenster, 1969). These genetic, cytological, biochemical, and biophysical studies all strongly support the current view that heterochromatin represents repressed segments of chromosomal DNA while euchromatin represents active segments, and that these two physical conformations of DNA can be interconvertible, and are an expression of the degree of nuclear differentiation within individual differentiated cells (Nakatsu et al, 1972; Frenster and Herstein, 1973).
II. Ultrastructure of Heterochromatin and Euchromatin
A wide variety of cellular processes involve de-repression of previously repressed genes (Table I),
and cells undergoing such gene de-repression often display
a reversible transformation of heterochromatin to euchromatin within their
nuclei (Figs. 1 and 2).
One of the most striking of such transformations occurs during the in vitro activation of peripheral blood lymphocytes by phytohemagglutinin (Tokuyasu et al, 1968). During such blastic transformation, the interphase lymphocyte nucleus enlarges two- to four-fold; heterochromatin (Fig. 1) is largely converted to euchromatin (Fig.2); the nucleolus undergoes a very marked hypertrophy, associated with a conversion of cytoplasmic monoribosomes to polysomes, and correlating with an extensive new synthesis of ribosomal and messenger RNA in such lymphocytes (Cooper, 1968). Electron microscopic autoradiography of lymphocytes undergoing blastic transformation by tritiated phytohemagglutinin has revealed that phytohemagglutinin is largely localized to the heterochromatin portions of the nucleus, resulting in an early conversion of such heterochromatin to euchromatin (Stanley et al, 1971).
This transformation of heterochromatin to euchromatin, with concurrent gene de-repression and accelerated RNA synthesis, is completely reversible if the activating phytohemagglutinin is removed from the incubation mixture (Polgar et al, 1968). Under these circumstances, euchromatin is transformed to heterochromatin within these interphase lymphocytes and RNA synthesis declines.
Another cell process in which a similar transformation is seen is during normal cell mitosis (Fig. 3 ). With the onset of prophase, the nuclear membrane undergoes dissolution, and the previous euchromatin of the interphase cell is now condensed into large chromosomal masses in preparation for the segregation and separation of the chromosomes later in metaphase. Strictly speaking, such condensation of interphase euchromatin into condensed chromosomal masses during prophase cannot be viewed as a transformation to heterochromatin (this term is reserved for interphase cells) (Heitz, 1929). Aside from the obvious ultrastructural similarity to heterochromatin, it is interesting to note that a comparable decrease is noted in the rate of RNA synthesis within such condensed mitotic chromosomes (Ohno and Makino, 1961; Taylor, 1960) as is noted within condensed heterochromatin during cell interphase (Frenster et al, 1963; Simmons et al, 1973).
During cell differentiation and maturation, RNA synthesis declines (Feinendegan et al, 1964), and is accompanied by a corresponding conversion of euchromatin to heterochromatin (Grasso et al, 1962) with a decreasing number of available DNA template sites for RNA synthesis (Nakatsu et al, 1972; Frenster and Herstein, 1973).
When nuclei are isolated under isotonic conditions from interphase cells, the relations between heterochromatin and euchromatin are well preserved (Fig. 4). These isolated nuclei are also still capable of RNA synthesis (Frenster et al, 1963) that is typical of the tissue of origin (Paul and Gilmour, 1968). If such nuclei are further extracted under hypotonic conditions (Frenster et al, 1963) in order to extract nuclear ribonucleoprotein particles and saline-soluble proteins (Frenster et al, 1960), the nuclei are then observed to swell to more than twice their normal size, allowing a clear definition of the relations between heterochromatin and euchromatin (Fig. 5).
At higher magnifications of such swollen interphase nuclei (Fig. 6), extended euchromatin fibrils can be seen to be ultrastructurally continuous with more condensed fibrils within heterochromatin, with a sharp zone of transition that is often less than 10 nm of euchromatin fibril length (Frenster, 1965a). In these preparations, the average caliber of euchromatin fibrils is seen to be 10 nm; and on occasion, single euchromatin fibrils can be followed for up to 1.0 um of their length (Frenster, 1965a).
III. Functions of Heterochromatin and Euchromatin:
It has become possible to separate heterochromatin from euchromatin after isolation of interphase cell nuclei (Frenster et al, 1963). Such preparations of isolated heterochromatin and euchromatin have permitted a wide variety of biochemical and biophysical studies which have delineated both the composition and the functions of heterochromatin and euchromatin (Frenster, 1965b).
When isolated nuclei were first incubated with RNA precursors before the separation of heterochromatin from euchromatin, it was found that euchromatin contained contined the majority of newly synthesized RNA (Frenster et al, 1963) and this distribution was subsequently confirmed by electron microscopic autoradiography (Littau et al, 1964). Compositional analysis of isolated heterochromatin and euchromatin revealed their equal content of histones in relationship to DNA, but an excess of nuclear polyanions such as RNA, residual proteins, and phosphoproteins in the euchromatin (Frenster, 1965b). If such nuclear polyanions, found in excess within isolated euchromatin, were added to isolated heterochromatin, a de-repression of RNA synthesis within heterochromatin was observed (Frenster, 1965b), suggesting a normal physiological role in control of gene transcription for these nuclear polyanions.
These macromolecular relationships were formalized in a model of
gene de-repression (Frenster, 1965c) in which
polyanionic nuclear ligands such as de-repressor RNA effect partial displacement
of repressor histones from particular portions of the DNA genome, allowing
DNA strand separations and the synthesis of messenger RNA species on such
de-repressed sites (Fig.7):
Fig. 7. Macromolecular interactions within heterochromatin
and euchromatin (Frenster, 1965c). Within
repressed heterochromatin, polycationic histones are tightly bound to DNA
templates, stabilizing such templates against strand separations and gene
transcription (Frenster, 1965b). Within active
euchromatin, histone repressors had been displaced from DNA templates by
polyanionic de-repressors such as de-repressor RNA (Frenster,
1965b) allowing DNA strand separations and gene transcription.
The model of gene de-repression (Fig. 7) also suggested that nuclear ligands might be expected to have varying effects on RNA synthesis according to their preference for binding to either single- or double-stranded DNA (Frenster, 1965e). When a large number of nuclear ligands are examined (Table II):
it can be seen that a striking correlation does indeed exist in
which ligands with a preference for double-stranded DNA are all seen to
inhibit RNA synthesis (Frenster, 1965e) by
decreasing the likelihood of localized DNA strand separations prior to
transcription, while ligands with a preference for single-stranded DNA
conversely increase RNA synthesis (Frenster,
1965e) by enhancing the likelihood of DNA strand separations prior
to transcription.
Such localized DNA strand separations have been detected within isolated euchromatin but not within isolated heterochromatin (Frenster, 1965d), and and have been recently proposed as a general feature of all RNA synthesis on DNA templates Crick, 1971).
When isolated interphase nuclei were first incubated with DNA precursors before separation of heterochromatin from euchromatin, it was found that euchromatin contained the majority of newly synthesized DNA (Frenster et al, 1963), confirming the earlier demonstration of the distribution by the technique of autoradiography (Hay and Revel, 1963). It was subsequently shown that the exact chromosomal segments which were transcribed for RNA synthesis in the G1 phase of interphase were the same segments that underwent earliest DNA synthesis in the S phase of interphase (Klevecz and Hsu, 1964), suggesting that DNA template states which allowed the synthesis of RNA were similarly favorable for the subsequent synthesis of DNA (Frenster, 1965d).
Conversely, the recognition that heterochromatin replicates its DNA late in the S phase of interphase (Lima-De-Faria, 1969; Grumbach et al, 1963) has permitted a convenient identification of heterochromatin through the entire cell cycle, including that within metaphase chromosomes (German, 1964; Schmid, 1963).
The finding that histone repressors within isolated euchromatin are partially displaced from their underlying templates (Frenster, 1965b) has suggested that molecular probes with a high affinity for DNA in its isolated state might preferentially localize within the euchromatin of an intact cell nucleus, and thus be useful for localizing such DNA templates within intact cells.
Using acridine orange as a highly specific molecular probe for DNA
free of any overlying histones, it has become possible to visualize DNA
template sites by high-resolution electron microscopy (Frenster,
1971). This technique has been applied to living cells (Fig.
8) and to intact cells after glutaraldehyde fixation
(Fig. 9), permitting a quantitative
estimate of the number of DNA template sites per cell through the course
of bone marrow granulocytic or erythrocytic differentiation (Nakatsu
et
al, 1972). It has also been useful in assessing the degree of gene
de-repression among the various immune cells opposing neoplastic cell invasion
of intact lymph nodes (Fig. 10) (Frenster
and Herstein, 1973).
Somatic cell genetic analysis has revealed that all but one of the
X chromosomes in adult mammalian cells are repressed, each repressed X
chromosome forming distinct heterochromatic Barr sex chromatin bodies (Grumbach
et
al, 1963). The decision regarding which single X chromosome will remain
active is made early in embryonic life, and is the maintained with high
fidelity through more than 50 generations of daughter cells (Davidson
et
al, 1963). Not only are these control mechanisms sensitive to the number
of X chromosomes per cell, but perhaps also to the gene content of each
X chromosome (Grumbach et al, 1963), suggesting that feedback mechanisms
are reflecting the rate of synthesis of messenger RNA synthesis within
euchromatin in determining which X chromosome will be heterochromatinized
(Frenster, 1966). Recent data concerning the molecular mechanisms of messenger
RNA synthesis within euchromatin indicate that the immediate transcription
product RNA may include double-stranded RNA regions (Jelinek and Darnell,
1972) corresponding to duplexes between de-repressor RNA and operator RNA
(Herstein and Frenster, 1972), which would
serve to inhibit gene transcription by removing de-repressor RNA from active
DNA templates when rates of transcription at particular genes are excessive
(Fig. 11):
Fig. 11. Postulated feedback inhibition of de-repression at a single
gene within euchromatin. De-repressor RNA (dRNA) is thought to bind to
the anti-coding strand (anti-o) of the operator gene, allowing synthesis
of the transcription product RNA on the operator (o) and and the structural
gene (sg) DNA templates (Herstein and Frenster,
1972). Excessive rates of transcription would result in increased amounts
of operator RNA (oRNA) via cleavage from the transcription product RNA.
This would tend to decrease further transcription by removal of DRNA from
the DNA template via formation of homometric or heterometric duplex RNA
between dRNA and either oRNA or transcription product RNA , respectively.
Messenger RNA (mRNA) might be included as part of the heterometric duplex
RNA (Frenster and Herstein, 1973; Jelinek and Darnell,
1972).
This mechanism appears operative in non-mammalian systems
as well (Brown and Chandra, 1973).
The recognition that nuclear DNA can exist within at least two basic chromatin conformations has permitted a greater understanding of gene controls within individual cells in higher organisms. These basic epigenetic controls are affected by a variety of smaller nuclear molecules (Table II) which serve to modulate earlier chromatin differentiation occurring during the course of cell differentiation. In addition, of course, cytoplasmic influences on nuclear structure and function (Fig. 12) are profound, and are as yet nt understood. It is likely, however, that they too impinge on the basic chromatin mechanisms for controlling the ultrastructure and function of nuclear DNA.
This work was supported in part by Grants CA-10174 and AM-01006 from the National Institutes of Health and by a Research Scholar Award from the Leukemia Society.
Archibald RB, and Frenster JH, (1973), Monogr. Nat. Cancer Inst. 36: 239.
Brown SW, and Chandra HS, (1973), Proc. Natl. Acad. Sci. U.S.A. 70: 195.
Brown SW, and Nur U, (1964), Science 145: 130.
Cooper HL, (1968), J. Biol. Chem. 243: 34.
Cooper KW, (1959), Chromosoma 10: 535.
Crick F, (1971), Nature 234: 25.
Dahmus ME, and Bonner J, (1970), Fed. Proc. 29: 1255.
Davidson RG, Nitowsky HM, and Childs B, (1963), Proc. Natl. Acad. Sci. U.S.A. 50: 481.
Feinendegan LE, Bond VP, Cronkite EP, and Hughes WL, (1964), Ann. N.Y. Acad. Sci. 113: 727.
Frenster JH, (1965a), Nature 205: 1341.
Frenster JH, (1965b), Nature 206: 680.
Frenster JH, (1965c), Nature 206: 1269.
Frenster JH, (1965d), Nature 208: 894.
Frenster JH, (1965e), Nature 208: 1093.
Frenster JH, (1966), in: "The Cell Nucleus", (Klouwen HM, ed.), pp. 27-49, Taylor and Francis, London.
Frenster JH, (1969), in: "Handbook of Molecular Cytology", (Lima-De-Faria A, ed.), pp. 251-276, North-Holland Publ., Amsterdam.
Frenster JH, (1971), Cancer Res. 31: 1128.
Frenster JH, (1972), Nature 236: 175.
Frenster JH, and Rogoway WM, (1970), Proc. 5th. Annu. Leukocyte Culture Conf., (Harris JE, ed.), pp. 359-371, Academic Press, New York.
Frenster JH, and Herstein PR, (1973), New Eng. J. Med. 288: 1224.
Frenster JH, Allfrey VG, and Mirsky AE, (1960), Proc. Natl. Acad. Sci. U.S.A., 46: 432.
Frenster JH, Allfrey VG, and Mirsky AE, (1963), Proc. Natl. Acad. Sci. U.S.A., 50: 1026.
German JL, (1964), J. Cell Biol. 20: 37.
Grasso JA, Swift H, and Ackerman GA, (1962), J. Cell Biol. 14: 235.
Grumbach MM, Morishima A, and Taylor JH, (1963), Proc. Natl. Acad. Sci. U.S.A., 49: 581.
Hay ED, and Revel JP, (1963), J. Cell Biol. 16: 29.
Heitz E, (1929), Ber. Deut. Bot. Ges. 47: 274.
Herstein PR, and Frenster JH, (1972), In: "Embryonic and Fetal Antigens in Cancer", (Anderson NG, and Coggin JH, eds.), Vol. 2, pp. 5-7, Natl. Tech. Informat. Service, U.S. Dept. Commerce, Springfield, Virginia.
Hess O, and Meyer GF, (1963), J. Cell Biol. 16: 527.
Holmes DS, Mayfield JE, Sander G, and Bonner J, (1972), Science 177: 72.
Hsu TC, (1962), Exp. Cell Res. 27: 332.
Jelinek W, and Darnell JE, (1972) Proc. Natl. Acad. Sci. U.S.A., 69: 2537.
Klevecz RR, and Hsu TC, (1964), Proc. Natl. Acad. Sci. U.S.A., 52: 811.
Lima-De-Faria A, (1969), In: "Handbook of Molecular Cytology", (Lima-De-Faria A, ed.), pp. 278-325, North-Holland Publ., Amsterdam.
Littau VC, Allfrey VG, Frenster JH, and Mirsky AE, (1964), Proc. Natl. Acad. Sci. U.S.A., 52: 93.
Lyon MF, (1961), Nature 190: 372.
McConaughy BL, and McCarthy BJ, (1972), Biochemistry 11: 998.
Nakatsu SL, Landrum S, and Frenster JH, (1972), Blood 40: 944.
Ohno S, and Makino S, (1961), Lancet 1: 78.
Paul J, and Gilmour RS, (1968), J. Mol. Biol. 34: 305.
Polgar PR, Kibrick S, and Foster JM, (1968), Nature 218: 596.
Schmid W, (1963), Cytogenetics 2: 175.
Simmons T, Heywood P, and Lodge L, (1973), J. Cell Biol. 59: 150.
Stanley DA, Frenster JH, and Rigas DA, (1971), Proc. 4th Annu. Leukocyte Culture Conf. (McIntyre OR. ed.), pp. 1-11, Appleton, New York.
Taylor JH, (1960), Ann. N. Y. Acad. Sci. 90: 409.
Tokuyasu K, Madden SC, and Zeldis LJ, (1968), J. Cell Biol. 39: 630.
"Electron Microscopy of Human Lymphocytes before and after Activation by PHA", (Busch, 1974).
Electron Micrographs for: "Ultrastucture and Function of Heterochromatin and Euchromatin".
Busch H, "The Cell Nucleus", Vols. 1-3, 1974, Academic Press, New York.
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