Prof. John H. Frenster

Laboratory of Cell Biology, Rockefeller Institute, New York.

Up to 80 percent of the DNA of interphase calf thymus lymphocytes is inactive as a template for synthesis of RNA (1), such inactivity being due to a repression mechanism which is itself sensitive to trypsin digestion (2). Polycationic histone proteins extracted from lymphocyte nuclei are capable of repressing the template function of DNA in a variety of cell-free systems (3-5), and such histones are sensitive to trypsin digestion (2). These results suggest that histones may function as repressors of DNA template function within the cells of higher organisms (6), but the mechanisms which overcome such histone repression are as yet poorly understood (7).

Repressed chromatin and chromatin active in RNA synthesis can each be isolated from interphase calf thymus lymphocytes (8). Investigations utilizing labelling with RNA precursors before chromatin isolation (8) or before electron microscopic autoradiography (9) have each demonstrated that repressed chromatin occurs as condensed masses of heterochromatin while active chromatin occurs as extended microfibrils of euchromatin (8). The extended microfibrils are ultrastructurally continuous with the condensed masses (8, 10), and up to 80 percent of the DNA of these interphase lymphocytes can be recovered in the repressed chromatin fraction (8). Furthermore, recent investigations of the preen gland in ducks disclose that when testosterone is used to stimulate nuclear RNA synthesis, both labelled testosterone and newly synthesized RNA are localized within the active chromatin fraction (11).

When the DNA is isolated from the repressed and from the active chromatin fractions of calf thymus lymphocytes of the same animal (12), using the fractionation and analytical methods previously described (8, 13), no significant differences in either the average base composition (14) or in the thermal hyperchromicity (Tm) (15) of the DNAs from the two chromatin fractions are found (Fig. 1). By contrast, when the whole chromatin fractions are studied (15) without first isolating their DNA (Fig. 1), the Tm of DNA within active chromatin (81^o C.) is significantly less than that of DNA within repressed chromatin (86^o C.)

Similarly, when human anti-DNA antibodies are used in an agar immunodiffusion study (16), the DNA within repressed chromatin is not reactive with the antibodies, while both the DNA within active chromatin and the DNAs isolated from either repressed or active chromatin are fully reactive (17). Histones are known to form electrostatic comlexes with DNA which result in increased stability of the DNA to thermal denaturation (15, 18) and in reduced reactivity of the DNA to anti-DNA antibodies (17). Since repressed and active chromatin contains near-equal amounts of histones (Table 1), these data suggest that the DNA within active chromatin is less firmly complexed by its histones than is the DNA within repressed chromatin (7).

When both the total histones and the lysine-rich histones are extracted (19) from active and repressed chromatin isolated from the same animal (8), the total histone contents of the two chromatin fractions relative to the DNA contents are found to be not significantly different (Table 1), with 20 percent of the total histones within each chromatin fraction being of the lysine-rich variety. Electrophoresis of the total histones on cellulose polyacetate (20) reveals no significant difference 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 types and quantities of histones found previously within animal cells varying widely in their tissue of origin (21), age (22), rate of RNA synthesis (22), or neoplastic character (23, 24), and suggests that alteration of the types or quantities of histones does not account for the differences in RNA synthetic activity found between active and repressed chromatin (7).
 
 
 

Table 1. Relation of Nuclear Constituents to DNA within Isolated Chromatin Fractions.

	No. of Animals	Active Chromatin (mg/100 mg DNA)	Repressed Chromatin (mg/100 mg DNA)	Active/repressed
Total histones	10	90.7 +/- 7.7	101.1 +/- 9.1	0.90 +/- 0.07
Non-histone  residual proteins	4	109.0 +/- 6.1	54.9 +/- 5.7	2.00 +/- 0.08
Total  phospholipids	5	17.0 +/- 3.6	3.7 +/- 1.3	4.93 +/- 1.26
Total ribonucleic acids	8	9.0 +/- 4.7	1.8 +/- 0.7	5.15 +/- 2.86
Total phospho- protein phosphorous	4	0.418 +/- 0.028	0.117 +/- 0.019	3.74 +/- 0.56
Mean +/- S.E.

By contrast, when the nuclear polyanion contents of active and repressed chromatin fractions prepared from the same animal (8) are determined relative to the DNA contents (Table 1), active chromatin is found to contain a two-fold excess of total non-histone residual proteins remaining after extractions of histones (25-28), a five-fold excess of total RNA and of total phospholipids (29), and an almost four-fold excess of total phosphoprotein phosphorus (30). Such nuclear phosphoproteins (30-32) constitute up to 15 percent of the non-histone residual proteins (30). Previous studies have suggested the importance of the non-histone residual proteins in the metabolism of chromatin (25-28) and within certain neoplasms (33). A parallel increase in both non-histone proteins and in RNA has recently been found in the soluble chromatin of active tissues in the chicken (22), in the active puffs of polytene chromosomes (34), and in the active loops of lampbrush chromosomes (35). The presence of these nuclear polyanions in excess within active chromatin suggests that they may play a part in antagonizing the DNA-histone interaction within active chromatin (7).

When active and repressed chromatin fractions are isolated from the same animal (8) and washed once in 0.25 M sucrose-0.010 M MnCl₂ to avoid subsequent gelation, the synthesis of RNA can then be studied in the isolated state. In such experiments, the chromatin fractions contained both the RNA polymerase (22, 36, 37) and the template DNA (22, 36, 37) needed for such synthesis, and were suspended as equal concentrations of either active chromatin or repressed chromatin (4-8 mg. DNA/ml.) from the same animal in an incubation medium of the following composition: 0.1 M tris-hydroxymethylaminomethane HCl, pH 7.7, 0.005 M MgCl₂, 0.002 M MnCl₂, 0.04 M NaF, 0.002 M GTP, CTP, and either ATP or UTP, in an incubation volume of 2.0 ml./flask, to which was added 0.5 uC. uridine-2-¹⁴C-triphosphate (spec. act. 21.0 mC./mM) or 0.5 uC. adenosine -8-¹⁴C-triphosphate (spec.act. 30.9 mC./mM). The flasks were shaken in air at 37^o C. for up to 30 min., the reaction was stopped by the addition of 5.0 ml. of ice-cold 10 percent trichloroacetic acid, and the DNA, RNA, protein, and incorporated radioactivity determined as previously described (13).

In such incubations of isolated chromatin fractions (Fig. 2), the extended active chromatin fraction remained 3-8 times as active in RNA synthesis as compared to the condensed repressed cchromatin fraction of the same animal, suggesting that the mechanisms controlling RNA synthesis within the intact nucleus have survived the isolation procedure, and are still functioning within these isolated chromatin fractions (7).
 
 
 

Table 2. Effect of Added Enzymes and Inhibitors on ATP-8-¹⁴C Incorporation into RNA of Isolated Chromatin Fractions.

	Isolated active chromatin (c.p.m./mg DNA)	Isolated repressed chromatin (c.p.m./mg DNA
Control	51.7 +/- 1.1	29.1 +/- 1.7
DNase	25.6 +/- 0.2	14.3 +/- 0.7
RNase	38.5 +/- 2.1	19.1 +/- 0.6
Actinomycin	30.1 +/- 0.1	15.4 +/- 0.3
Puromycin	65.2 +/- 3.1	28.3 +/- 0.4
Trypsin	163.0 +/- 16.8	104.6 +/- 3.5
Mean +/- S.E.

That the observed incorporation of UTP-2-¹⁴C or ATP-8-¹⁴C represents RNA synthesis is shown by the following experiments (Table 2):
(a). the radioactive product of incorporation is insoluble in cold 5 percent trichloroacetic acid, and 70-90 percent of the incorporated radioactivity is removed on subsequent digestion with RNase;
(b). the incorporation process is sensitive to small amounts (100 ug/mg DNA) of actinomycin D (38), or to the addition (1.0 mg/ml.) of DNase or RNase, and requires the simultaneous presence of all four ribonucleoside triphosphates.
In these properties the RNA synthetic process is similar to that demonstrated earlier within other chromatin preparations isolated from animal tissues (22, 36).

The addition of trypsin to incubations of intact nuclei results in a selective removal of histones by the preferential hydrolysis of peptide bonds involving lysine and arginine, effecting marked increases in the rate of nuclear RNA synthesis (2). Similar additions of trypsin (1.0 mg/ml.) to incubations of isolated chromatin fractions result in a marked increase in RNA synthesis within both isolated active and repressed chromatin (Table 2), indicating that active chromatin is partially repressed by its constituent histones, and that both forms of chromatin can increase their rates of RNA synthesis following removal of their histones. When the synthetic polyanion polyethylene sulphonate is added to incubations of isolated active and repressed chromatin fractions of the same animal (Fig. 3), a marked increase in RNA synthesis occurs within each chromatin fraction, rivalling in magnitude the increase in RNA synthesis produced by the removal of histones during trypsin digestion (Table 2). Polyethylene sulfonate is a polyanion of high molecular weight and charge density (39), and lacks any of the modifying basic and/or hydrophobic side groups that characterize the naturally occurring nuclear polyanions (Table 1).

The total RNA of both the nuclear and cytoplasmic fractions were each isolated from the calf thymus lymphocytes of a single animal by the phenol extraction procedure using an aqueous phase consisting of 0.1 M tris HCl, pH 8.5 (40), and collecting only that RNA which is precipitated from solution by 10 percent sodium chloride and which is free of any contaminating DNA. A comparable preparation of yeast RNA (41) was obtained from the Worthington Biochemical Corp. The addition of thymus total nuclear RNA to incubations of active or repressed chromatin fractions of the same animal results in an increase in the synthetic activity within isolated repressed chromatin (Fig. 3), but has little effect on active chromatin, while the addition of yeast RNA actually decreases the synthetic activity of isolated active chromatin (Fig.3).
 
 
 

Table 3. Effect of Added Nuclear Constituents on UTP-2-¹⁴C Incorporation into RNA of Isolated Chromatin Fractions.

	Isolated active chromatin (c.p.m./mg DNA)	Isolated repressed chromatin (c.p.m./mg DNA)
Control	222.2 +/- 13.8	76.2 +/- 4.4
Total nuclear RNA	249.5 +/- 8.5	185.2 +/- 11.8
Soluble protein fraction	262.0 +/- 1.0	121.7 +/- 0.3
Non-histone residual proteins	286.0 +/- 1.0	118.4 +/- 7.2
Phosphatidylcholine micelles	265.5 +/- 9.5	93.6 +/- 0.4
Total histones	97.6 +/- 4.8	59.9 +/- 4.8
Mean +/- S.E.

When several of the major classes of nuclear polyanions which are found in excess within active chromatin are each tested by addition (1.0 mg nuclear polyanion/mg chromatin DNA) to incubation of isolated active or repressed chromatin (Table 3), little or no effect is observed on the rate of RNA synthesis within active chromatin, but RNA synthesis within repressed chromatin is significantly increased. In the case of added nuclear RNA, this increase raises the level of synthesis within repressed chromatin almost to the basal synthetic level of active chromatin (Table 3). Addition of isolated nuclear phosphoprotein (30) similarly results in two-fold increases in the rate of RNA synthesis within isolated repressed chromatin. Nuclear RNA, non-histone residual proteins (24), and phosphoproteins (30) are all polyanions which are slightly modified by the presence of basic and/or hydrophobic side groups in their molecular structure. The water-soluble micelles of phospholipids which were tested (Table 3) also behave as modified polyanions (42), and polyanions have been demonstrated (43) in the soluble-nuclear fraction (13) which was tested. By contrast, the addition of lyophilized polycationic histones (19) at concentrations of 1.0 mg histone/mg chromatin DNA decreases RNA synthesis within each chromatin fraction (Table 3), decreasing the synthesis within active chromatin almost to the basal level of repressed chromatin.

When high molecular weight RNAs of various sources are compared by addition to incubations of isolated repressed and active chromatin (Fig. 4), it is seen that total nuclear RNA is significantly more effective than total cytoplasmic RNA prepared from the same calf thymus lymphocytes in increasing the synthetic activity within isolated repressed chromatin. The RNA extracted from thymus nuclear ribosomes (44) is less effective in this regard (Fig. 4), as is E. coli Ss-RNA stripped of its amino-acids (45) or high-molecular-weight yeast RNA (41).

These comparative RNA experiments suggest that total nuclear RNA of calf thymus lymphocytes contains a species of RNA that is particularly effective in increasing the synthetic activity within repressed chromatin. Since the phosphate content and hence the polyanionic character of all RNAs is generally uniform, such metabolic effectiveness may reside in the secondary structure or in the base sequence structure of this nuclear RNA species, which could enable it to hybridize with complementary sequences in specific portions of the repressed DNA genome (46), providing a mechanism for high specificity in such de-repression of RNA synthesis. The effectiveness of other nuclear polyanions as de-repressors of RNA synthesis (Table 3) may be less specific for particular portions of the DNA genome, and may be related to their interaction with steroid hormones (47) or polycyclic carcinogens (48, 49).

Acknowledgements:

I thank Drs. A.E. Mirsky, V.G. Allfrey, H.G. Rose, M.L. Green, E.M. Tan, T.A. Langan, and R. Faulkner for their advice.

Support:

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

1. Allfrey VG, and Mirsky AE, Proc. Natl. Acad. Sci. U.S. 48, 1590 (1962).
2. Allfrey VG, Littau VC, and Mirsky AE, Proc. Natl. Acad. Sci. U.S. 49, 414 (1963).
3. Frenster JH, Allfrey VG, and Mirsky AE, Biochim. Biophys. Acta 47, 130 (1961).
4. Allfrey VG, in Biological Structure and Function, edit. by Goodwin TW, Lindberg O, 1, 261 (Academic Press, New York, 1961).
5. Huang RC, and Bonner J, Proc. Natl. Acad. Sci. U.S. 48, 1216 (1962).
6. Allfrey VC, and Mirsky AE, in Synthesis and Structure of Macromolecules: Cold Spring Harbor Symp. Quant. Biol. 28, 247 (1963).
7. Frenster JH, J. Cell Biol. 23, 32A (1964).
8. Frenster JH, Allfrey VC, and Mirsky AE, Proc. Natl. Acad. Sci. U.S. 50, 1026 (1963).
9. Littau VC, Allfrey VG, Frenster JH, and Mirsky AE, Proc. Natl. Acad. Sci. U.S. 52, 93 (1964).
10. Frenster JH, Nature 205, 1341 (1965).
11. Loeb PM, and Wilson JD, Clin. Res. 13, 45 (1965).
12. Marmur J, J. Mol. Biol. 3, 208 (1961).
13. Frenster JH, Allfrey VG, and Mirsky AE, Proc. Natl. Acad. Sci. U.S. 46, 432 (1960).
14. Wyatt GR, and Cohen SS, Biochem. J. 55, 774 (1953).
15. Bonner J, and Huang RC, J. Mol. Biol. 6, 169 (1963).
16. Kunkel HG, and Tan EM, Adv. Immunol. 4, 351 (1964).
17. Tan EM, (in the press).
18. Allfrey VG, Faulkner R, and Mirsky AE, Proc. Natl. Acad. Sci. 51, 786 (1964).
19. Johns EW, and Butler JAV, Biochem. J. 82, 15 (1962).
20. Green ML, (in the press).
21. Phillips DMP, Prog. Biophys. and Biophys. Chem. 12, 211 (1962).
22. Dingman CW, and Sporn MB, J. Biol. Chem. 239, 3483 (1964).
23. Laurence DJ, Simpson P, and Butler JAV, Biochem. J. 87, 200 (1963).
24. Busch H, and Steele WJ, Adv. Cancer Res. 8, 41 (1964).
25. Mirsky AE, and Ris H, J. Gen. Physiol. 34, 475 (1951).
26. Daly MM, Allfrey VG, and Mirsky AE, J. Gen. Physiol. 36, 173 (1952)
27. Allfrey VG, Daly MM, and Mirsky AE, J. Gen. Physiol. 38, 415 (1955).
28. Allfrey VG, Mirsky AE, and Osawa S, J. Gen. Physiol. 40, 451 (1957).
29. Rose HG, and Frenster JH, Biochim. Biophys. Acta (in the press).
30. Langan TA, and Lipmann F, J. Biol. Chem. (in the press).
31. Johnson RM, and Albert S, J. Biol. Chem. 200, 335 (1953).
32. Sperti S, Lorini M, Pinna LA, and Moret V, Biochim. Biophys. Acta 82, 476 (1964).
33. Steele WJ, and Busch H, Cancer Res. 23, 1153 (1963).
34. Beermann W, Amer. Zoologist 3, 23 (1963).
35. Gall JG, and Callan HG, Proc. Natl. Acad. Sci. U.S. 48, 602 (1962).
36. Weiss SB, Proc. Natl. Acad. Sci. U.S. 46, 1020 (1960).
37. Chamberlin M, and Berg P, Proc. Natl. Acad. Sci. U.S. 48, 81 (1962).
38. Reich E, Franklin RM, Shatkin AJ, and Tatum EL, Proc. Natl. Acad. Sci. U.S. 48, 1238 (1962).
39. Allfrey VG, and Mirsky AE, Proc. Natl. Acad. Sci. U.S. 44, 981 (1958).
40. Brawerman G, Biochim. Biophys. Acta 76, 322 (1963).
41. Crestfield AM, Smith KC, and Allen FW, J. Biol. Chem. 216, 185 (1955).
42. Abramson AB, Katzman R, and Gregor HP, J. Biol. Chem. 239, 70 (1964).
43. Patel G, and Wang TY, Exp. Cell Res. 34, 120 (1964).
44. Sibatani A, deKloet SR, Allfrey VG, and Mirsky AE, Proc. Natl. Acad. Sci. U.S. 48, 471 (1962).
45. Tissieres A, Bourgeois S, and Gros F, J. Mol. Biol. 7, 100 (1963).
46. McCarthy BJ, and Hoyer BH, Proc. Natl. Acad. Sci. U.S. 52, 915 (1964).
47. Talwar GP, Segal SJ, Evans A, and Davidson OW, Proc. Natl. Acad. Sci. U.S. 52, 1059 (1964).
48. Loeb LA, and Gelboin HV, Proc. Natl. Acad. Sci. U.S. 52, 1219 (1964).
49. Bakay B, and Sorof S, Cancer Res. 24, 1814 (1964).

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