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The Structure of DNA in Native Chromatin as Determined by Ethidium Bromide Binding
The Structure of DNA in Native Chromatin as Determined by Ethidium Bromide Binding J. PAOLETTI,~ B. B. MAGEEAND P. T. MAGEE Department of Human Genetics Yale University School of Medicine New Haven, Connecticut
1. Introduction The currently favored model of chromatin structure was first suggested by the electron micrographs of Olins and Olins ( 1 ) and is supported by various biophysical and biochemical studies ( 2 4 ) .This model proposes that a “core,” containing two molecules each of histones 2a, 2b, 3 and 4, is spaced at 200-nucleotide distances along the DNA backbone. Thc fifth histone, H1, associates more loosely with the DNA a t some point as yet undeterrnincd, possibly between the cores or v-bodies (nucleosomes). Axel (5) has produced evidence that both active and inactive genes are present in the “-body fraction isolated from chromatin. The state of the DNA and the nature of its interaction with histones and nonhistone proteins has important implications for the mechanism ( s ) by which transcription of this highly organized structure is controlled. We chose to use the intercalating dye ethidium bromide to investigate this problem. Specifically, we wished to ask: what is the tertiary structure of the DNA on the v-bodies?
II. Methods Chromatin was prepared from WIL2 lymphoblastoid cell nuclei ( 6 ) according to No11 ( 7 ) with digestion times, and enzyme concentrations varied as described in the figure legends. Ethidium bromide binding was followed by fluorescence enhancement ( 8). Fluorescence polarization was determined on a spectrofluorimeter as described by Yguerabide et al. ( 9 ) . Binding data were plotted as a Scatchard plot, where T = ethidium bromide bound per nucleotide of DNA, and c = unbound Present address: Laboratorie de Pharmacologie MolCculaire no 147 du CNRS, Institut Gustave Roussy, 16bis Av Paul Vaillant Couturier, 94800 Villejuif, France. 373
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ethidium bromide ( 10). The fluorescence polarization data were plotted as cos?w (the square of the average cosine of the angle w swept by the emission oscillator between the time of absorption and emission of light) against T .
111. Results Figure 1 shows the Scatchard plot of ethidium bromide binding to free DNA, to minimally digested chromatin (30 seconds), and to extensively digested chromatin ( lo’), A number of very striking features are evident in the chromatin curves. First, the extrapolation to T / C = 0, a measure of the total amount of dye bound per nucleotide, is much less for both kinds of chromatin than for free DNA. The difference (0.110.12 vs. 0.20) could be characteristic of that found for circular DNA (11)
6
5
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1
0.05
0.1
r
0.2
FIC. 1. Scatchard plot of ethidium bromide binding to chromatin and DNA. Ethidiuni Iiromide binding to chromatin and DNA was detemiined as described ( 8 ) . The DNA standard was from calf thymus (Worthington) (-). Chromatin was prepared by digestion for 30 scconds with 1 pg of staphylucoecal nuclease (Worthington) per 1.5 x 10’ ririclei a t 37°C (0-0) or by 10 minutes’ digestion under ). The 30-second (minimally) digested chromatin had identical conditions ( A-A >1% monomer as determined by gel electrophoresis of the extracted DNA; the 10-minute (extensively) digested chromatin had 11% monoiner. [See Note Added in Proof, 1). 377.1
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or could correspond to a complete masking by proteins of one-half of the DNA, and has been found previously for chromatin extracted by mechanical shearing (12). Second, the binding to minimally digested chromatin falls into two classes, one with a high binding constant ( k = 2 X lo6 M-I), not too different from that for free DNA under these conditions (Fig. l ) , and one with a low binding constant ( k = 2 X ' loi M - I ) . The latter constant is very similar to that found for DNA in high salt, when the phosphate groups are largely neutralized. The region of greater affinity, while observable in extensively digested chromatin, has a much lower constant than in minimally digested chromatin. Two such classes were also found in sheared chromatin (12). An extrapolation of the high-affinity region to T / C = 0, while difficult owing to the curvature of the line, gives a value of about 0.03 to 0.05, corresponding to 2540%of the total DNA, or 50 to 80 base-pairs. This number is in fairly good agreement with the fraction of DNA in a v-body that is highly susceptible to nuclease digestion, 50-60 base-pairs. A third important characteristic of the Scatchard plot in Fig. 1 is the transition that separates the high- and low-affinity binding domains in minimally digested chromatin. This transition is typical of a cooperativity in binding. We interpret these results to mean that v-bodies do contain DNA in two different states, differing in their affinity for ethidium bromide (EtdBr). The fractions of DNA in these states corresponds reasonably well with the fractions in the nuclease-sensitive and nuclease-resistant parts of the v-body. Further support for this interpretation comes from the similarity between the affinity constant for EtdBr of the fraction corresponding to the limit digest of the DNA and the affinity constant of DNA in high salt, since DNA tightly complexed with histones is, so to speak, in high salt. The cooperativity of binding to the low affinity region may indicate that the unwinding associated with intercalation of the dye causes a partial relaxation of the structure so that further binding is facilitated. The fact that the total amount bound is characteristic of a closed circular configuration could argue that even in the partially relaxed structure the DNA of v-bodies still acts as though highly constrained. In order to substantiate further the interpretation that the highaffinity fraction of DNA is in a separate region from the low-affinity fraction, we looked at the polarization of fluorescence of EtdBr bound to chromatin. When EtdBr is excited with fully polarized light, the emitted light is partially depolarized. This depolarization occurs when the molecule can rotate during the lifetime (20 nsec) of the excited state or when the energy of excitation can be transferred to a molecule a t an angle with the first ( f o r example, a second EtdBr intercalated nearby). Thus the
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polarization of fluorescence extrapolated to low values of r gives a measure of the rigidity of the DNA into which the dye is intercalated, and the rate of depolarization as a function of r is proportional to the frequency with which molecules of dye intercalate in proximity. Figure 2 shows that in free DNA, we find (extrapolating to r = 0) a value of cos‘o= 0.835, while in chromatin the value is 1.0. If the polarization of EtdBr in DNA is extrapolated to r = 0 a t infinite viscosity, the value is very close to 1.0. (Infinite viscosity would prevent the rotation of the DNA around the long axis, accompanied by the transient opening or “breathing” of the base-pairs.) Thus, the DNA that binds the dye at very low r (therefore, the high affinity fraction) seems to be highly constrained and unable to undergo rotational motion. Figure 2 also shows that the depolarization of fluorescence in v-body DNA as r increases is very rapid. We interpret this to be due to rapid saturation of the highaffinity DNA with EtdBr, accompanied by energy transfer to nearby moleules. The possibility that the depolarization is due to energy transfer to externally bound dye, rather than to intercalated mdecules, seems to be eliminated by the fact that the lifetime of fluorescence is constant with increasing 7 ( 19 ns) and is very close to that of EtdBr in free DNA (20.7 ns). After r reaches about 0.03-0.035, the depolarization of fluorescence in v-bodies declines at a rate roughly parallel to that in free DNA, indicating that the dye molecules are able to intercalate over larger fractions of the DNA in the chromatin. The following ideas about the structure of v-bodies can be drawn from
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FIG. 2. Polarization of fluorescence of ethidium bromide bound to chromatin. The chromatin (O---O) was minimally digested as described for Fig. 1. The DNA ( 0-0 ) was native calf thymus ( Worthington).
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these studies: ( 1 ) DNA in v-bodies is heterogeneous, and both fractions seem to be constrained; ( 2 ) the fraction of DNA with high affinity for EtdBr corresponds in amount to the one that is highly sensitive to nuclease and that is decreased when digestion time is increased; ( 3 ) in order to bind ethidium bromide, the DNA fraction with low affinity must undergo some structural alterations. These ideas, leading as they do to a picture of chromatin with a highly organized and tightly constrained structure, indicate that transcription may be a very complex process, requiring a number of relaxing proteins or other structural entities to permit binding and progression of RNA polymerase.
ACKNOWLEDGMENTS This research was supported by USPHS grants GM 19481 ( E . A. Adelberg, principal investigator), and GM 21012 (P. T. Magee, principal investigator).
REFERENCES 1 . A. L. Olins and D. E. Olins, Science 183, 330 (1974). 2. K. E. van Holde, C. C . Sahasrabnddhe, B. R. Shaw, E. F. J. von Bruggen and H. Arnberg, BBRC 60, 1365 (1974). 3. D. R. Hewish and L. A. Burgoyne, BBRC 52, 504 ( 1973). 4. M. Noll, J. 0. Thomas and R. D. Kornberg, Science 187, 1203 (1975). 5. R. Axel, This volume, p. 355. 6. B. B. Magee, J. Paoletti and P. T. Magee, PNAS 72, 4830 (1975). 7. M. Noll, Natuw 251, 249 ( 1974). 8. J. B. Le Pecq and C . Paoletti. J M B 27, 87 (1967). 9. J. Yguerabide, H. F. Epstein and L. Stryer, J M B 51,573 (1970). 10. G. Scatchard, Ann. N.Y. Acad. Sci. 51, 660 (1949). 1 1 . J. B. Le Pecq, Methods Biochem. Anal. 20, 41 (1972). 12. L. M. Angerer, S. Georghiov and E. N. Mondrianakis, Bchem. 13, 1073 (1974).
NOTE ADDEDIN PROOF In later experiments we have found that the time of digestion, up to 10 minutes or 20%monomer, does not affect the Scatchard plot of ethidium bromide binding to nnclease-digested chromatin, We attribute the difference shown in Fig. 1 to a de-
gradation of the 10-minute digested chromatin, possibly due to protease action. This does not, of course, affect the conclusions drawn about the structure of nucleasedigested chromatin.
1. Introduction The currently favored model of chromatin structure was first suggested by the electron micrographs of Olins and Olins ( 1 ) and is supported by various biophysical and biochemical studies ( 2 4 ) .This model proposes that a “core,” containing two molecules each of histones 2a, 2b, 3 and 4, is spaced at 200-nucleotide distances along the DNA backbone. Thc fifth histone, H1, associates more loosely with the DNA a t some point as yet undeterrnincd, possibly between the cores or v-bodies (nucleosomes). Axel (5) has produced evidence that both active and inactive genes are present in the “-body fraction isolated from chromatin. The state of the DNA and the nature of its interaction with histones and nonhistone proteins has important implications for the mechanism ( s ) by which transcription of this highly organized structure is controlled. We chose to use the intercalating dye ethidium bromide to investigate this problem. Specifically, we wished to ask: what is the tertiary structure of the DNA on the v-bodies?
II. Methods Chromatin was prepared from WIL2 lymphoblastoid cell nuclei ( 6 ) according to No11 ( 7 ) with digestion times, and enzyme concentrations varied as described in the figure legends. Ethidium bromide binding was followed by fluorescence enhancement ( 8). Fluorescence polarization was determined on a spectrofluorimeter as described by Yguerabide et al. ( 9 ) . Binding data were plotted as a Scatchard plot, where T = ethidium bromide bound per nucleotide of DNA, and c = unbound Present address: Laboratorie de Pharmacologie MolCculaire no 147 du CNRS, Institut Gustave Roussy, 16bis Av Paul Vaillant Couturier, 94800 Villejuif, France. 373
374
J . PAOLE’ITI ET AL.
ethidium bromide ( 10). The fluorescence polarization data were plotted as cos?w (the square of the average cosine of the angle w swept by the emission oscillator between the time of absorption and emission of light) against T .
111. Results Figure 1 shows the Scatchard plot of ethidium bromide binding to free DNA, to minimally digested chromatin (30 seconds), and to extensively digested chromatin ( lo’), A number of very striking features are evident in the chromatin curves. First, the extrapolation to T / C = 0, a measure of the total amount of dye bound per nucleotide, is much less for both kinds of chromatin than for free DNA. The difference (0.110.12 vs. 0.20) could be characteristic of that found for circular DNA (11)
6
5
2
1
0.05
0.1
r
0.2
FIC. 1. Scatchard plot of ethidium bromide binding to chromatin and DNA. Ethidiuni Iiromide binding to chromatin and DNA was detemiined as described ( 8 ) . The DNA standard was from calf thymus (Worthington) (-). Chromatin was prepared by digestion for 30 scconds with 1 pg of staphylucoecal nuclease (Worthington) per 1.5 x 10’ ririclei a t 37°C (0-0) or by 10 minutes’ digestion under ). The 30-second (minimally) digested chromatin had identical conditions ( A-A >1% monomer as determined by gel electrophoresis of the extracted DNA; the 10-minute (extensively) digested chromatin had 11% monoiner. [See Note Added in Proof, 1). 377.1
ETHIDIUM BROMIDE BINDING TO CHROMATIN
375
or could correspond to a complete masking by proteins of one-half of the DNA, and has been found previously for chromatin extracted by mechanical shearing (12). Second, the binding to minimally digested chromatin falls into two classes, one with a high binding constant ( k = 2 X lo6 M-I), not too different from that for free DNA under these conditions (Fig. l ) , and one with a low binding constant ( k = 2 X ' loi M - I ) . The latter constant is very similar to that found for DNA in high salt, when the phosphate groups are largely neutralized. The region of greater affinity, while observable in extensively digested chromatin, has a much lower constant than in minimally digested chromatin. Two such classes were also found in sheared chromatin (12). An extrapolation of the high-affinity region to T / C = 0, while difficult owing to the curvature of the line, gives a value of about 0.03 to 0.05, corresponding to 2540%of the total DNA, or 50 to 80 base-pairs. This number is in fairly good agreement with the fraction of DNA in a v-body that is highly susceptible to nuclease digestion, 50-60 base-pairs. A third important characteristic of the Scatchard plot in Fig. 1 is the transition that separates the high- and low-affinity binding domains in minimally digested chromatin. This transition is typical of a cooperativity in binding. We interpret these results to mean that v-bodies do contain DNA in two different states, differing in their affinity for ethidium bromide (EtdBr). The fractions of DNA in these states corresponds reasonably well with the fractions in the nuclease-sensitive and nuclease-resistant parts of the v-body. Further support for this interpretation comes from the similarity between the affinity constant for EtdBr of the fraction corresponding to the limit digest of the DNA and the affinity constant of DNA in high salt, since DNA tightly complexed with histones is, so to speak, in high salt. The cooperativity of binding to the low affinity region may indicate that the unwinding associated with intercalation of the dye causes a partial relaxation of the structure so that further binding is facilitated. The fact that the total amount bound is characteristic of a closed circular configuration could argue that even in the partially relaxed structure the DNA of v-bodies still acts as though highly constrained. In order to substantiate further the interpretation that the highaffinity fraction of DNA is in a separate region from the low-affinity fraction, we looked at the polarization of fluorescence of EtdBr bound to chromatin. When EtdBr is excited with fully polarized light, the emitted light is partially depolarized. This depolarization occurs when the molecule can rotate during the lifetime (20 nsec) of the excited state or when the energy of excitation can be transferred to a molecule a t an angle with the first ( f o r example, a second EtdBr intercalated nearby). Thus the
376
J. PAOLETIT ET AL.
polarization of fluorescence extrapolated to low values of r gives a measure of the rigidity of the DNA into which the dye is intercalated, and the rate of depolarization as a function of r is proportional to the frequency with which molecules of dye intercalate in proximity. Figure 2 shows that in free DNA, we find (extrapolating to r = 0) a value of cos‘o= 0.835, while in chromatin the value is 1.0. If the polarization of EtdBr in DNA is extrapolated to r = 0 a t infinite viscosity, the value is very close to 1.0. (Infinite viscosity would prevent the rotation of the DNA around the long axis, accompanied by the transient opening or “breathing” of the base-pairs.) Thus, the DNA that binds the dye at very low r (therefore, the high affinity fraction) seems to be highly constrained and unable to undergo rotational motion. Figure 2 also shows that the depolarization of fluorescence in v-body DNA as r increases is very rapid. We interpret this to be due to rapid saturation of the highaffinity DNA with EtdBr, accompanied by energy transfer to nearby moleules. The possibility that the depolarization is due to energy transfer to externally bound dye, rather than to intercalated mdecules, seems to be eliminated by the fact that the lifetime of fluorescence is constant with increasing 7 ( 19 ns) and is very close to that of EtdBr in free DNA (20.7 ns). After r reaches about 0.03-0.035, the depolarization of fluorescence in v-bodies declines at a rate roughly parallel to that in free DNA, indicating that the dye molecules are able to intercalate over larger fractions of the DNA in the chromatin. The following ideas about the structure of v-bodies can be drawn from
1.0
0.5
-
I
0.01
I
I
0.05
r
0.1
FIG. 2. Polarization of fluorescence of ethidium bromide bound to chromatin. The chromatin (O---O) was minimally digested as described for Fig. 1. The DNA ( 0-0 ) was native calf thymus ( Worthington).
ETHIDIUM BROMIDE BINDING TO CHROMATIN
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these studies: ( 1 ) DNA in v-bodies is heterogeneous, and both fractions seem to be constrained; ( 2 ) the fraction of DNA with high affinity for EtdBr corresponds in amount to the one that is highly sensitive to nuclease and that is decreased when digestion time is increased; ( 3 ) in order to bind ethidium bromide, the DNA fraction with low affinity must undergo some structural alterations. These ideas, leading as they do to a picture of chromatin with a highly organized and tightly constrained structure, indicate that transcription may be a very complex process, requiring a number of relaxing proteins or other structural entities to permit binding and progression of RNA polymerase.
ACKNOWLEDGMENTS This research was supported by USPHS grants GM 19481 ( E . A. Adelberg, principal investigator), and GM 21012 (P. T. Magee, principal investigator).
REFERENCES 1 . A. L. Olins and D. E. Olins, Science 183, 330 (1974). 2. K. E. van Holde, C. C . Sahasrabnddhe, B. R. Shaw, E. F. J. von Bruggen and H. Arnberg, BBRC 60, 1365 (1974). 3. D. R. Hewish and L. A. Burgoyne, BBRC 52, 504 ( 1973). 4. M. Noll, J. 0. Thomas and R. D. Kornberg, Science 187, 1203 (1975). 5. R. Axel, This volume, p. 355. 6. B. B. Magee, J. Paoletti and P. T. Magee, PNAS 72, 4830 (1975). 7. M. Noll, Natuw 251, 249 ( 1974). 8. J. B. Le Pecq and C . Paoletti. J M B 27, 87 (1967). 9. J. Yguerabide, H. F. Epstein and L. Stryer, J M B 51,573 (1970). 10. G. Scatchard, Ann. N.Y. Acad. Sci. 51, 660 (1949). 1 1 . J. B. Le Pecq, Methods Biochem. Anal. 20, 41 (1972). 12. L. M. Angerer, S. Georghiov and E. N. Mondrianakis, Bchem. 13, 1073 (1974).
NOTE ADDEDIN PROOF In later experiments we have found that the time of digestion, up to 10 minutes or 20%monomer, does not affect the Scatchard plot of ethidium bromide binding to nnclease-digested chromatin, We attribute the difference shown in Fig. 1 to a de-
gradation of the 10-minute digested chromatin, possibly due to protease action. This does not, of course, affect the conclusions drawn about the structure of nucleasedigested chromatin.