Molec. Biol. Rep. Vol. 4, 2:97-100
CHANGES IN NONHISTONE CHROMOSOMAL PROTEINS IN PHYTOHEMAGGLLrrININSTIMULATED LYMPHOCYTES
Horst BLUTHMANN
Max-Planck-Institut fiir V~rusforschung, Tfibingen, German Fed. Republic (Received December 22, 19 77, revised version January 30, 19 78)
Abstract
Materials and methods
Stimulation of bovine lymphocytes with phytohemagglutinin results in quantitative as well as qualitative changes in the nonhistone chromosomal proteins. Analysis of these proteins by hydroxyapatite chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis shows not only a selective increase in the amount of some nonhistone proteins but also a decrease of other nonhistone protein bands. This observation is compatible with the view that nonhistone proteins have an inhibitory as well as an activating function at the genome level.
Lymphocyte cell culture
Introduction
Preparation of nuclei and chromatin
In numerous systems, in which cellular differentiation can be followed, a quantitative change in the amount of nonhistone chromosomal proteins has been shown to be an early event. An increase in the amount or synthesis of nonhistone proteins has been consistently reported to accompany the increase in metabolic activity of the cell. In the specific case of lymphocytes, stimulation by plant mitogens is followed by an enhanced synthesis (8, 10, 14) or increase in amount (10) of nuclear nonhistone proteins, beside other changes which include a stimulation of histone acetylation (15) and nuclear protein phosphorylation (11). The gen regulatory function of nonhistone proteins is compatible not only with an increase in the amount of activating nonhistone proteins but also with a decrease of inhibitory ones. A concomitant increase and decrease of specific nonhistone chromosomal proteins in phytohemagglutinin stimulated lymphocytes is shown in this study in which nonhistone fractions were analysed by hydroxyapatite chromatography and polyacrylamide gel electrophoresis.
Isolation of nuclei and chromatin were as described previously (2, 4).
Bovine lymphocyte cultures were prepared as described by Hausen et al. (7). The cells were maintained in Eagle's medium supplemented with 10% foetal calf serum at a concentration of 10 7 cells/ml. All lymphocytes were preincubated for 20 h prior to experimental use. 500 ml of the culture were stimulated for I h, 4 h, 48 h and 64 h, respectively with 0.5 ml phytohemagglutinin P (Difco).
Fractionation of nonhistone proteins on hydroxyapatite Chromosomal proteins were fractionated on hydroxyapatite columns into two histone and four nonhistone protein fractions as described previously (3).
Polyacrylamide gel electrophoresis Discontinuous gel electrophoresis was performed according to Laemmli (12) in 0.56 x 10 cm tubes using 5% stacking gels and 12.5% sample gels. 0.10.25 ml samples containing 30 ~tg nonhistone proteins were dialysed against 0.06 M Tris pH 6.8, 2% sodium dodecylsulfate, 10% glycerol and 5% 2-mercaptoethanol and heated for 1.5 min in boiling water prior to electrophoresis. Approximate molecular weights were calibrated against the relative migrations ( 1 8 ) o f bovine serum albumin (67,000) and dimer (135,000), 97
ovalbumin (45,000), chymotrypsinogen (25,000), and myoglobin (17,800).
Analyses Chromatin was analysed for its components as described elsewhere (4).
Results and discussion
In Table 1 the amounts of histone and nonhistone chromosomal proteins in resting lymphocytes and in cells which were stimulated for 1 h, 4 h, 48 h and 64 h by the addition of phytohemagglutinin are compared. Whereas the histone content remains almost unchanged, the amount of chromosomal nonhistone proteins increases slightly after 4 h and is enhanced by a factor of 1.7 after 48 h. A 64 h period of stimulation does not further increase the nonhistone protein content. Table 1. Analysesof chromatin from lymphocytesat various times after stimulation. Time of stimulation (h) 0 1 4 48 64
Histone Nonhistone protein % 103 109 100 110 103
21 21 (0) 26 (23) 37 (76) 36 (71)
RNA
1.6 2.7 (69) 2.6 (63) 3.8 (140) 3.7 (130)
Values are given as percentages relative to the DNA content. Percent stimulation in relation to the unstimulated level is given between brackets. An increase in amount of nuclear acidic proteins early after induction of lymphocyte proliferation has been reported by Johnson et al. (10). Within 15 min after addition of the mitogen Concanavalin A a 1.3fold increase was detected as a result of an influx of presynthesized proteins from the cytoplasm to the nucleus. In this study, however, an increase in the amount of chromosomal nonhistone proteins is not detected until 4 h after stimulation. This difference may result from the fact that the different chromatin isolation procedure used in this study excludes loosely bound nonhistone proteins extractable from chromatin by 0.3 M sodium chloride. Table 1 also includes data on the amount of chromatin associated RNA during the course of activation. As early as 1 h after the addition of the mitogen, the content of chromatin RNA goes up by a factor of 1.7 and is more than trebled after 48 h. On longer stimulation 98
this value seems to stay constant. This RNA may contain metabolically active as welt as structural RNA. It is possible that some species of chromosomal RNA may play a role in this process of gene activation by modulating the general structure of chromatin. A more specific function of this RNA, namely as DNA sequence specific binding elements, has been proposed by Bekhor et al. (1) and Huang & Huang (9), and is compatible with current models of gene regulation (5, 6). Besides quantitative changes in the amount of nonhistone chromosomal proteins during induction of proliferation, qualitative changes in the pattern of these proteins were also analysed in this study. To this end chromatin was prepared from lymphocytes that had been stimulated for various times. The nonhistone proteins were separated by hydroxyapatite into four fractions and analysed on sodium dodecylsulfate polyacrylamide gels. As shown earlier (3), the relative amounts of nonhistone proteins in the four hydroxyapatite fractions are 55%, 20%, 10% and 15%, respectively. These ratios do not change during stimulation. Figure 1 a-d shows the result of this comparative study. The gel scans immediately reveal that several qualitative as well as quantitative changes occur during stimulation with phytohemagglutinin. Only striking changes are discussed here. Early after induction, that is during the first hour after addition of the mitogen, a group of proteins in fractions NH3 and NH4 ranging in molecular weight from 40,000 to 110,000 becomes more prominent relative to the main peak of MW 30,000 in these fractions (Fig. lc and ld). Since the amount of total nonhistone protein is not changed at this time (see Table 1), the higher molecular weight species must increase in amount at the expense of the main peak. The amount of the higher molecular weight peptides still increase at later times in the activation process, concomitant with an increase in the amount of total nonhistone proteins. Figure la shows that 64 h after induction another group of nonhistone proteins, namely those in fraction NH1, is greatly enhanced in relative amounts. These are proteins with molecular weights of 22,000, 24,000, 30,000 and 38,000, respectively as indicated by the arrows. Whether these proteins are involved in DNA synthesis and mitosis, a process which is activated 2-3 days after the addition of phytohemagglutinin, has not been investigated. A reduction of individual protein bands is also evident. For instance, a 40,000 dalton species in fraction NH3 is virtually absent at later stages of induction (Fig. lc). The same is true for a 80,000
A)
Oh
Oh
B)
I
I Ii
i i
lh
i I
Oh
I
I I
Oh D)
c)
lh
lh
[!: '4h ~
4h
4h
4h
64h ,
I 111'
~
.
'-J
L
100 70
50
30
20
MOLWT It 10 .3
10
I~
70
50
30
20
10
MOLWT.xlO-3
1(30 ?0
50
30
20
MOLWT x 10-3
10
I(]O
70
SO
30
20
tO
MOL-WT, x 10-3
Fig. 1. Polyacrylamide gel electrophoresis of hydroxyapatite nonhistone fractions of unstimulated lymphocytes (0 h) and 1 h, 4 h and 64 h, respectively, after stimulation. (A) Nonhistone protein fraction NH1; (B) NH2; (C) NH3; (D) NH4.
dalton species in fraction NH4 (Fig. ld). Moreover, in fraction NH2 two polypeptides with a molecular weight of approximately 100,000 and 110,000 are reduced 64 h after induction (Fig. lb). Upon closer examination other changes in the polypeptide patterns can also be detected. The purpose of this analysis, however, is to show that not only an increase in the amount of individual nonhistone proteins bands can be observed after induction of lymphocytes but also a decrease in the amount of some polypeptides, some of which are virtually absent at later stages in this process of differentiation. Double isotop labeling experiments by other workers (8, 14, 16, 17)have always shown a stimulatory effect of plant mitogens on the synthesis of nuclear acidic proteins. A selective reduction of the amount of individual nonhistone proteins upon
activation of resting lymphocytes has not been reported previously. However, it is compatible with the role of nonhistone proteins as controlling elements in the regulation of gene activation to have inhibitory as well as activating functions. Hence in an activation process such as the transformation of a resting cell to proliferation a reduction of inhibitory molecules may occur. This is exactly what is found in this study underlining the possible involvement of nonhistone proteins in both the activation and repression of genetic information.
Acknowledgments The author is indebted to Heinz Pachowsky and Alfred Sch6ffski for their excellent assistance.
99
References 1. Bekhor, I., G.M. Kung & J. Bonner. J. Mol. Biol. 39: 351-364 (1969). 2. Bliithmann, H. Eur. J. Biochem. 70:233-240 (1976). 3. Bliithmann, H., S. Mrozek & A. Gierer. Eur. J. Biochem. 58:315-326 (1975). 4. Bliithmann, H. Int. J. Biochem. (in press). 5. Davidson, E.H. & R.J. Britten. Q. Rev. Biol. 48: 565613 (1973). 6. Gierer, A. Cold Spring Harbor Symp. Quant. Biol. 38: 951-961 (1973). 7. Hausen, P., H. Stein & H. Peters. Eur. J. Biochem. 9: 542-549 (1969). 8. Hemminki, K. Exptl. Cell Res. 9 3 : 6 3 - 7 0 (1975). 9. Huang, R.C. & P.C. Huang (1969). J. Mol. Biol. 39: 365-378 (1969). 10. Johnson, E.M., J. Karn & V.G. Allfrey. J. Biol. Chem. 249:4990-4999 (1974). 11. Kleinsmith, L.J., V.G. Allfrey & A.E. Mirsky. Science 154:780-781 (1966). 12. Laemmli, U.K. Nature 227:680-685 (1970). 13. Levy, R., S. Levy, S.A. Rosenberg & R.T. Simpson. Biochemistry 12:224-228 (1973). 14. Levy, R. & S.A. Rosenberg. J. lmmunol. 108: 10731079 (1972). 15. Pogo, B.G.T., V.G. Allfrey & A.E. Mirski. Proc. Natl. Acad. Sci. USA 55:805-812 (1966). 16. Renaud, J., M.C. Eggo & W.I. Waithe. Ann. Immunol. (Inst. Pasteur) 128C: 909-913 (1977). 17. Rosenberg, S. & R. Levyt J. Immunol. 108:1105-1109 (1972). 18. Shapiro, A.L., E. Vinuela & J.V. Maizel. Biochem. Biophys. Res. Comm. 28:815-820 (1967).
100
CHANGES IN NONHISTONE CHROMOSOMAL PROTEINS IN PHYTOHEMAGGLLrrININSTIMULATED LYMPHOCYTES
Horst BLUTHMANN
Max-Planck-Institut fiir V~rusforschung, Tfibingen, German Fed. Republic (Received December 22, 19 77, revised version January 30, 19 78)
Abstract
Materials and methods
Stimulation of bovine lymphocytes with phytohemagglutinin results in quantitative as well as qualitative changes in the nonhistone chromosomal proteins. Analysis of these proteins by hydroxyapatite chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis shows not only a selective increase in the amount of some nonhistone proteins but also a decrease of other nonhistone protein bands. This observation is compatible with the view that nonhistone proteins have an inhibitory as well as an activating function at the genome level.
Lymphocyte cell culture
Introduction
Preparation of nuclei and chromatin
In numerous systems, in which cellular differentiation can be followed, a quantitative change in the amount of nonhistone chromosomal proteins has been shown to be an early event. An increase in the amount or synthesis of nonhistone proteins has been consistently reported to accompany the increase in metabolic activity of the cell. In the specific case of lymphocytes, stimulation by plant mitogens is followed by an enhanced synthesis (8, 10, 14) or increase in amount (10) of nuclear nonhistone proteins, beside other changes which include a stimulation of histone acetylation (15) and nuclear protein phosphorylation (11). The gen regulatory function of nonhistone proteins is compatible not only with an increase in the amount of activating nonhistone proteins but also with a decrease of inhibitory ones. A concomitant increase and decrease of specific nonhistone chromosomal proteins in phytohemagglutinin stimulated lymphocytes is shown in this study in which nonhistone fractions were analysed by hydroxyapatite chromatography and polyacrylamide gel electrophoresis.
Isolation of nuclei and chromatin were as described previously (2, 4).
Bovine lymphocyte cultures were prepared as described by Hausen et al. (7). The cells were maintained in Eagle's medium supplemented with 10% foetal calf serum at a concentration of 10 7 cells/ml. All lymphocytes were preincubated for 20 h prior to experimental use. 500 ml of the culture were stimulated for I h, 4 h, 48 h and 64 h, respectively with 0.5 ml phytohemagglutinin P (Difco).
Fractionation of nonhistone proteins on hydroxyapatite Chromosomal proteins were fractionated on hydroxyapatite columns into two histone and four nonhistone protein fractions as described previously (3).
Polyacrylamide gel electrophoresis Discontinuous gel electrophoresis was performed according to Laemmli (12) in 0.56 x 10 cm tubes using 5% stacking gels and 12.5% sample gels. 0.10.25 ml samples containing 30 ~tg nonhistone proteins were dialysed against 0.06 M Tris pH 6.8, 2% sodium dodecylsulfate, 10% glycerol and 5% 2-mercaptoethanol and heated for 1.5 min in boiling water prior to electrophoresis. Approximate molecular weights were calibrated against the relative migrations ( 1 8 ) o f bovine serum albumin (67,000) and dimer (135,000), 97
ovalbumin (45,000), chymotrypsinogen (25,000), and myoglobin (17,800).
Analyses Chromatin was analysed for its components as described elsewhere (4).
Results and discussion
In Table 1 the amounts of histone and nonhistone chromosomal proteins in resting lymphocytes and in cells which were stimulated for 1 h, 4 h, 48 h and 64 h by the addition of phytohemagglutinin are compared. Whereas the histone content remains almost unchanged, the amount of chromosomal nonhistone proteins increases slightly after 4 h and is enhanced by a factor of 1.7 after 48 h. A 64 h period of stimulation does not further increase the nonhistone protein content. Table 1. Analysesof chromatin from lymphocytesat various times after stimulation. Time of stimulation (h) 0 1 4 48 64
Histone Nonhistone protein % 103 109 100 110 103
21 21 (0) 26 (23) 37 (76) 36 (71)
RNA
1.6 2.7 (69) 2.6 (63) 3.8 (140) 3.7 (130)
Values are given as percentages relative to the DNA content. Percent stimulation in relation to the unstimulated level is given between brackets. An increase in amount of nuclear acidic proteins early after induction of lymphocyte proliferation has been reported by Johnson et al. (10). Within 15 min after addition of the mitogen Concanavalin A a 1.3fold increase was detected as a result of an influx of presynthesized proteins from the cytoplasm to the nucleus. In this study, however, an increase in the amount of chromosomal nonhistone proteins is not detected until 4 h after stimulation. This difference may result from the fact that the different chromatin isolation procedure used in this study excludes loosely bound nonhistone proteins extractable from chromatin by 0.3 M sodium chloride. Table 1 also includes data on the amount of chromatin associated RNA during the course of activation. As early as 1 h after the addition of the mitogen, the content of chromatin RNA goes up by a factor of 1.7 and is more than trebled after 48 h. On longer stimulation 98
this value seems to stay constant. This RNA may contain metabolically active as welt as structural RNA. It is possible that some species of chromosomal RNA may play a role in this process of gene activation by modulating the general structure of chromatin. A more specific function of this RNA, namely as DNA sequence specific binding elements, has been proposed by Bekhor et al. (1) and Huang & Huang (9), and is compatible with current models of gene regulation (5, 6). Besides quantitative changes in the amount of nonhistone chromosomal proteins during induction of proliferation, qualitative changes in the pattern of these proteins were also analysed in this study. To this end chromatin was prepared from lymphocytes that had been stimulated for various times. The nonhistone proteins were separated by hydroxyapatite into four fractions and analysed on sodium dodecylsulfate polyacrylamide gels. As shown earlier (3), the relative amounts of nonhistone proteins in the four hydroxyapatite fractions are 55%, 20%, 10% and 15%, respectively. These ratios do not change during stimulation. Figure 1 a-d shows the result of this comparative study. The gel scans immediately reveal that several qualitative as well as quantitative changes occur during stimulation with phytohemagglutinin. Only striking changes are discussed here. Early after induction, that is during the first hour after addition of the mitogen, a group of proteins in fractions NH3 and NH4 ranging in molecular weight from 40,000 to 110,000 becomes more prominent relative to the main peak of MW 30,000 in these fractions (Fig. lc and ld). Since the amount of total nonhistone protein is not changed at this time (see Table 1), the higher molecular weight species must increase in amount at the expense of the main peak. The amount of the higher molecular weight peptides still increase at later times in the activation process, concomitant with an increase in the amount of total nonhistone proteins. Figure la shows that 64 h after induction another group of nonhistone proteins, namely those in fraction NH1, is greatly enhanced in relative amounts. These are proteins with molecular weights of 22,000, 24,000, 30,000 and 38,000, respectively as indicated by the arrows. Whether these proteins are involved in DNA synthesis and mitosis, a process which is activated 2-3 days after the addition of phytohemagglutinin, has not been investigated. A reduction of individual protein bands is also evident. For instance, a 40,000 dalton species in fraction NH3 is virtually absent at later stages of induction (Fig. lc). The same is true for a 80,000
A)
Oh
Oh
B)
I
I Ii
i i
lh
i I
Oh
I
I I
Oh D)
c)
lh
lh
[!: '4h ~
4h
4h
4h
64h ,
I 111'
~
.
'-J
L
100 70
50
30
20
MOLWT It 10 .3
10
I~
70
50
30
20
10
MOLWT.xlO-3
1(30 ?0
50
30
20
MOLWT x 10-3
10
I(]O
70
SO
30
20
tO
MOL-WT, x 10-3
Fig. 1. Polyacrylamide gel electrophoresis of hydroxyapatite nonhistone fractions of unstimulated lymphocytes (0 h) and 1 h, 4 h and 64 h, respectively, after stimulation. (A) Nonhistone protein fraction NH1; (B) NH2; (C) NH3; (D) NH4.
dalton species in fraction NH4 (Fig. ld). Moreover, in fraction NH2 two polypeptides with a molecular weight of approximately 100,000 and 110,000 are reduced 64 h after induction (Fig. lb). Upon closer examination other changes in the polypeptide patterns can also be detected. The purpose of this analysis, however, is to show that not only an increase in the amount of individual nonhistone proteins bands can be observed after induction of lymphocytes but also a decrease in the amount of some polypeptides, some of which are virtually absent at later stages in this process of differentiation. Double isotop labeling experiments by other workers (8, 14, 16, 17)have always shown a stimulatory effect of plant mitogens on the synthesis of nuclear acidic proteins. A selective reduction of the amount of individual nonhistone proteins upon
activation of resting lymphocytes has not been reported previously. However, it is compatible with the role of nonhistone proteins as controlling elements in the regulation of gene activation to have inhibitory as well as activating functions. Hence in an activation process such as the transformation of a resting cell to proliferation a reduction of inhibitory molecules may occur. This is exactly what is found in this study underlining the possible involvement of nonhistone proteins in both the activation and repression of genetic information.
Acknowledgments The author is indebted to Heinz Pachowsky and Alfred Sch6ffski for their excellent assistance.
99
References 1. Bekhor, I., G.M. Kung & J. Bonner. J. Mol. Biol. 39: 351-364 (1969). 2. Bliithmann, H. Eur. J. Biochem. 70:233-240 (1976). 3. Bliithmann, H., S. Mrozek & A. Gierer. Eur. J. Biochem. 58:315-326 (1975). 4. Bliithmann, H. Int. J. Biochem. (in press). 5. Davidson, E.H. & R.J. Britten. Q. Rev. Biol. 48: 565613 (1973). 6. Gierer, A. Cold Spring Harbor Symp. Quant. Biol. 38: 951-961 (1973). 7. Hausen, P., H. Stein & H. Peters. Eur. J. Biochem. 9: 542-549 (1969). 8. Hemminki, K. Exptl. Cell Res. 9 3 : 6 3 - 7 0 (1975). 9. Huang, R.C. & P.C. Huang (1969). J. Mol. Biol. 39: 365-378 (1969). 10. Johnson, E.M., J. Karn & V.G. Allfrey. J. Biol. Chem. 249:4990-4999 (1974). 11. Kleinsmith, L.J., V.G. Allfrey & A.E. Mirsky. Science 154:780-781 (1966). 12. Laemmli, U.K. Nature 227:680-685 (1970). 13. Levy, R., S. Levy, S.A. Rosenberg & R.T. Simpson. Biochemistry 12:224-228 (1973). 14. Levy, R. & S.A. Rosenberg. J. lmmunol. 108: 10731079 (1972). 15. Pogo, B.G.T., V.G. Allfrey & A.E. Mirski. Proc. Natl. Acad. Sci. USA 55:805-812 (1966). 16. Renaud, J., M.C. Eggo & W.I. Waithe. Ann. Immunol. (Inst. Pasteur) 128C: 909-913 (1977). 17. Rosenberg, S. & R. Levyt J. Immunol. 108:1105-1109 (1972). 18. Shapiro, A.L., E. Vinuela & J.V. Maizel. Biochem. Biophys. Res. Comm. 28:815-820 (1967).
100
