Volume 4 Number 4 April 1977
Nucleic Acids Research
Salt-induced structural changes in nucleosomes
C.G.Sahasrabuddhe and Grady F. Saunders The Department of Developmental Therapeutics, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, TX 77030, USA Received 13 January 1977
ABSTRACT
Nucleosomes and oligonucleosomes were prepared by digestion of human placental nuclei with staphlococccal nuclease and fractionated by gel filtration chromatography. The effect of increasing salt on the structure of nucleosomes Nucleosomes and was examined in the presence and absence of 10 mM MqCl oligonucleosomes are insoluble over a broad range of sil t concentration. Nucleosomes are soluble in >120 mM (NH4) S04 containing 10 mM MgCl allowing analyses of changes in nucleosomal DNA b« C.D. spectroscopy. Nucleosomes are insoluble in < 120 mM (NH ) SO containing 10 mM MgCl as demonstrated by turbidity measurements. We cancluie that the insolubiliit of nucleosomes accompanies salt-induced structural changes possibly due to individual particle condensation. As the salt concentration is increased the nucleosomes condense and then relax at higher salt concentrations.
INTRODUCTION
Eukaryotic chromatin exists as a regularly repeating structure that resembles beads on a string. The repeating subunits, nucleosomes, contain 140-150 base pairs and are connected by 50-60 base pairs of the continuing double-stranded DNA (1-11). The DNA in the nucleosome appears to be tightly folded around a protein core of 8 histone molecules ( 2 each of H2A, H2B, H3, and H4) (12-14). The interaction between the histone core and DNA is not sequence specific (8,15-18). Some evidence has been obtained that DNA contained in nucleosomes is transcribed in vivo (19-20). One approach to examining the accessibility of nucleosomal DNA for transcription involves the use of DNA-dependent RNA polymerase in vitro as a chemical probe of chromatin structure. Recently we have obtained evidence for in vitro transcribability of nucleosomes (21). RNA polymerase requires both divalent (Mg ++or Mn ) and monoor K ) cations for activity (22). Therefore, it is important to valent ( (NH4)4
~~~ ++ ~~~~~++
know the effect of salt and divalent cation (Mg or Mn ) on the template, chromatin or nucleosomes,for this enzyme (21,23). Structural changes are induced in purified histones by salt and the salt
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
853
Nucleic Acids Research concentration has a profound effect on the kinetics of complex formation among histones (24-28). Since histone octamers are present in the nucleosome cores, one would expect to see the induction of structural changes in these octamers in the presence of salt. The DNA in the nucleosome is very closely associated with the histone octamer core; hence the structural changes in the octamer should also cause corresponding structural changes in nucleosomal DNA. These changes can be easily detected by analyses of the circular dichroism spectra.Alterations in the structure of DNA in the nucleosome resulting from salt-induced structural changes in the histone core can be detected by measurement in the 260-320 nm region of the C.D. spectrum since histones do not contribute to the spectrum in this region. MATERIALS AND METHODS Isolation of Nucleosomes Fresh human pl acenta was cut i nto smal l pi eces and the pi eces washed wi th i cecold SSC (0.15 M NaCl, 0.015 M Na3citrate) 3 times. The washed placental pieces were immediately frozen on dry ice and stored at -200C until used. Forty grams of frozen placenta were quickly thawed and homogenized in a Waring blender in a medium containing SSC, 5 mM NaHS03, and 0.2% NP-40 (Shell Oil Co.). The homogenate was filtered once through 8 layers of cheesecloth and twice through 2 layers of Miracloth (Chicopee Mills, Inc.). The filtrate was centrifuged at 1200 x g for 15 min to obtain the crude nuclear pellet. The nuclear pellet was washed once with SSC, once with 0.5 X SSC, and once with phosphate buffer (5 mM sodium phosphate, pH 6.8, containing 0.1 mM CaC12). The fi nal pel l et was suspended i n 3-5 ml of phosphate buffer. Staphyl ococcal nucl ease (Worthington Biochemical Corp., NFCP grade) was added to this suspension at a concentration of 10 units/A260 and the mixture was incubated at 370C for 12 min. At this time about 35-40% of the original amountofnucleic acid was made soluble in 5% trichloroacetic acid. The reaction was stopped by adding 0.1 M EDTAto make a final concentration of 5 mM and cooling the reaction mixture in an ice bath. The reaction mixture was dialysed against 10 mM Tris-HCl buffer, pH 7.8, containing 0.2 mM EDTA (Tris-EDTA buffer). The nuclear debris and the insoluble particulate material were removed by centrifuging the mixture at 12,000 x g for 30 riin. The clear supernatant contained nucleosomes and oligonucleosomes. Fractionation of Nucleosomes The nucleosome preparation(20-30 A260in 2 ml)was applied toa Bio-Gel A-5m column(llOcm x 1 .6cm)previously equilibrated with Tris-EDTA buffer (29). Flow rate was maintained at 5ml/cm2/hr and 2-mlfractions were collected.Nucleosomes and oligonucleosomes were obtained bypooling appropriatefractionsfrom the column. 854
Nucleic Acids Research Turbidity Measutements
Five microliters of either 2 M(NH4)2S04 or 2 M KC1 were added successively to the nucleosomal solution in Tris-EDTA buffer and the absorbance at 420 nm was monitored after each addtion. Turbidity was also measured in the presence of 10 mMM MgCl2. Circular Dichroism Measurements The circular dichroism spectra were measured on a Durrum Jasco CD-SP spectrophotometer at room temperature. The nucleosome concentration in each sample was such that the equivalent DNA concentration was always in the range of 50-80 ig/ml (similar to the one usedin chromatin transcription reactions) (30-32). The samples were prepared by mixing appropriate amounts of 1 M MgCl 29 2 M (NH4)2S04, or 2 M KC1 and stock solutions of nucleosomes and diluting the mixture with Tris-EDTA buffer. The value of {t}, the molar ellipticity, was calculated at 2-nm intervals, and plotted against wavelength. The DNA concentration in the nucleosomeswas determined using E260=6600' (3,6).
Polyacrylamide Gel Electrophoresis Three percent composite cylindrical gels(2.5% acrylamide(19:1, acrylamide: bis-acrylamide) 0.5% agarose)were employed to analyze the pooled nucleosomes from the Bio-Gelcolumn.The length of DNAfrom nucleosomes or oligonucleosomes was determined on 5.5% composite cylindrical gels(5%acrylamide (19: 1, acrylamide: bis-acrylamide) and 0.5% agarose) (10). Restriction fragments of PM2 DNA generated by restriction enzyme Hae III were run on a parallel gel as markers. Bromophenol blue was used to indicate the extent of migration on the gel. The running buffer was 40 mM Tris-HCl, pH 7.8, 20 mM sodium acetate, and 3 mM EDTA. The gels were run at a constant current of 2 mA per tube. The gels were stained in 2 lag/ml ethidium bromide solution in running buffer for 30 min and scanned on a Aminco Bowmen fluorospectrophotometer.The exciting wavelength was 510 nm and the fluorescence was measured at 590 nm.
Isolation of DNA The DNA from nucleosomes and oligonucleosomes was purified by phenol extraction (33). Phenol was redistilled and saturated with 0.01 M Tris-HCl, pH 7.8, containing 0.2 M NaCl and 0.2% 8-hydroxyquinoline, before use.
RESULTS Nucleosomes and Oligonucleosomes Nucleosomes and oligonucleosomes prepared by digestion of human placental 855
Nucleic Acids Research nuclei with staphylococcal nuclease were fractionated by gel filtration (Bio-Gel A-5m) chromatography. The elution profile (Fig. 1) can be resolved into four Gaussian curves. Pool I (fractions 63-68) contains more than 90% mononucleosomes with less than 10% contamination of dinucleosomes. If only the trailing side of the peak is used(fractions 66-70),there is no dinucleosome contamination. When the material from pool I is analyzed by electrophoresis on a 3% composite gel, a peak of nucleosomes and a small contaminating peak of dinucleosomes (Fig. 2) is observed. On the other hand, the material in pool II (fractions 40-58) contains mainly dinucleosomes and trinucleosomes with smaller amounts of mononucleosomes and tetranucleosomes (Fig. 2). Nucleosomes were analyzed on SDS-urea polyacrylamide(12.5%)gels and nucleosomal proteins stained with Coomasie blue.These gels show the presence of all 4histones invo'vedin the nucleosomal core as well as histone Hl. The ratio of the amount of HI to the amount of H4 suggests that >70% of the nucleosomes do not have Hl. The DNAs extracted from pools I and II were run on 5.5% composite cylindrical gels (Fig. 3). The repeat length of DNAis calculated tobe 185base pairs and the length of DNA
Gel Filtration (A-5m) Column of Nuclear Digest.
1.4 ~~~~l0~~~~~ E
°) 1.0
-
Pdl
t
OIimh_deommss
_-I
C>O.6
0.2 40
60 Fraction Number
80
Purified nuclei from human placenta of staphylococcal nuclease. The nuclear anAuWe clear supernatant fractionated on a Bio-
Figure 1. Gel filtration of the nuclear digest. were digested for 12 min with 10 units/A debris was removed by centrifugation
Gel A-5m column.
856
Nucleic Acids Research involved in the core is about 155 base pairs. Both these values compare well with the values for nucleosomal and core DNAs obtained from various tissues (10,1 1) . Turbidity Measurements The amount of insoluble or aggregated macromolecules in a suspension can be estimated from the absorbance at 420 nm. We have used this property to determine the effectsof salt concentrationon the solubilityof nucleosomesand oligonucleosomes. The turbidity measurements done on the nucleosome solutions indicate that the nucleosomes are partially insoluble over a wide range of {(NH4)2S04}(Fig. 4).There is a peak of turbidity at about 50 mM (NH4)2S04 for nucleosomes, while a broad peak in the range of 70-120 mM (NH4)2S04is observed for oligonucleosomes. Similarly, chromatin is partially insoluble over a broad range of NaCl concentrations (34). In order to examine the nature of this insolubility, we measured the effect of nucleosome concentration on turbidity (Fig. 5) in 50 mM (NH4)2S04. The linear relationship between nucleosome concentration and insolubility suggests that the state of aggregation of nucleosomes remains the same in the range of nucleosome concentration studied here.
Gel Electrophoresis of Nucleosomes and Oligonucleosomes. 2.5 % Acry lamide - 0.5 %/o Agarose Mono-
Pool I
Bottom
|
i -nuc le osomes ~~~~~Tr
Top
Figure 2. Analysis of nucleosomes by polyacrylamide gel electrophoresis. Pool I (fractions 63-68) and pool 11 (fractions 40-58) were run on 3. 0%6 composite cylindrical gels. The gels were stained with 2 ,g/ml ethidium bromide solution in running buffer and scanned with a fluorospectrophotometer. Fluorescence intensity is plotted versus migration distance.
857
Nucleic Acids Research Gel Electrophoresis of DNA from Nucleosomes and Oligonucleosomes (5% Acrylamide-0.5%Agarose) A.PM-2 DNA
HoellIdigest
592
288 ~n642
160
B. Pool I DNA
l5gl\-l~~~~~85
o
155
i
_LL
C. Pool IIDNA
410 Trimer
Monomer
Bottom
-
Migration
1610
Top
Figure 3. Length of nucleosomal DNA. Ihe DNA purified by phenol extraction of material from pools I and II was electrophoresed on 5. 5% composite cylindrical gels. The fluorescent scans were obtained after staining the gels with ethidium bromide. (A) PM-2 DNA fragments generated with Hae III restriction enzyme, used as markers. (B) DNA from material in pool I. (C) DNA from material in pool II. Migration was from right to left.
Effect of Salt on Turbidity of Nucleosomes and Oligonucleosomes
I cw
I @0N
-2
m
,0
[(NH4k2 S04]in mM Figure 4. Effect of (NH4) SO on the turbidity of nucleosomes and oligonucleosomes. To a nucleosome solution 60-A0 Mg) in Tris buffer 5 , I aliquots of 2 M (NH4) S04 solution Were added and mixed; the absorbance at 420 nm was read after each addition. ( o -o -) nucleosome (pool I) at a concentration of 60 ,Ag. (- *- oligonucleosomes (pool II) at a concentration of 70 Mg/ml. 858
)
Nucleic Acids Research Effect of Nucleosome Concentration on Turbidity in 0.05 M [(NH4)2 S04]
0.14 0.12 E
0.10
¢ 0.08 -
0.06
Nucleic Acids Research
Salt-induced structural changes in nucleosomes
C.G.Sahasrabuddhe and Grady F. Saunders The Department of Developmental Therapeutics, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, TX 77030, USA Received 13 January 1977
ABSTRACT
Nucleosomes and oligonucleosomes were prepared by digestion of human placental nuclei with staphlococccal nuclease and fractionated by gel filtration chromatography. The effect of increasing salt on the structure of nucleosomes Nucleosomes and was examined in the presence and absence of 10 mM MqCl oligonucleosomes are insoluble over a broad range of sil t concentration. Nucleosomes are soluble in >120 mM (NH4) S04 containing 10 mM MgCl allowing analyses of changes in nucleosomal DNA b« C.D. spectroscopy. Nucleosomes are insoluble in < 120 mM (NH ) SO containing 10 mM MgCl as demonstrated by turbidity measurements. We cancluie that the insolubiliit of nucleosomes accompanies salt-induced structural changes possibly due to individual particle condensation. As the salt concentration is increased the nucleosomes condense and then relax at higher salt concentrations.
INTRODUCTION
Eukaryotic chromatin exists as a regularly repeating structure that resembles beads on a string. The repeating subunits, nucleosomes, contain 140-150 base pairs and are connected by 50-60 base pairs of the continuing double-stranded DNA (1-11). The DNA in the nucleosome appears to be tightly folded around a protein core of 8 histone molecules ( 2 each of H2A, H2B, H3, and H4) (12-14). The interaction between the histone core and DNA is not sequence specific (8,15-18). Some evidence has been obtained that DNA contained in nucleosomes is transcribed in vivo (19-20). One approach to examining the accessibility of nucleosomal DNA for transcription involves the use of DNA-dependent RNA polymerase in vitro as a chemical probe of chromatin structure. Recently we have obtained evidence for in vitro transcribability of nucleosomes (21). RNA polymerase requires both divalent (Mg ++or Mn ) and monoor K ) cations for activity (22). Therefore, it is important to valent ( (NH4)4
~~~ ++ ~~~~~++
know the effect of salt and divalent cation (Mg or Mn ) on the template, chromatin or nucleosomes,for this enzyme (21,23). Structural changes are induced in purified histones by salt and the salt
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
853
Nucleic Acids Research concentration has a profound effect on the kinetics of complex formation among histones (24-28). Since histone octamers are present in the nucleosome cores, one would expect to see the induction of structural changes in these octamers in the presence of salt. The DNA in the nucleosome is very closely associated with the histone octamer core; hence the structural changes in the octamer should also cause corresponding structural changes in nucleosomal DNA. These changes can be easily detected by analyses of the circular dichroism spectra.Alterations in the structure of DNA in the nucleosome resulting from salt-induced structural changes in the histone core can be detected by measurement in the 260-320 nm region of the C.D. spectrum since histones do not contribute to the spectrum in this region. MATERIALS AND METHODS Isolation of Nucleosomes Fresh human pl acenta was cut i nto smal l pi eces and the pi eces washed wi th i cecold SSC (0.15 M NaCl, 0.015 M Na3citrate) 3 times. The washed placental pieces were immediately frozen on dry ice and stored at -200C until used. Forty grams of frozen placenta were quickly thawed and homogenized in a Waring blender in a medium containing SSC, 5 mM NaHS03, and 0.2% NP-40 (Shell Oil Co.). The homogenate was filtered once through 8 layers of cheesecloth and twice through 2 layers of Miracloth (Chicopee Mills, Inc.). The filtrate was centrifuged at 1200 x g for 15 min to obtain the crude nuclear pellet. The nuclear pellet was washed once with SSC, once with 0.5 X SSC, and once with phosphate buffer (5 mM sodium phosphate, pH 6.8, containing 0.1 mM CaC12). The fi nal pel l et was suspended i n 3-5 ml of phosphate buffer. Staphyl ococcal nucl ease (Worthington Biochemical Corp., NFCP grade) was added to this suspension at a concentration of 10 units/A260 and the mixture was incubated at 370C for 12 min. At this time about 35-40% of the original amountofnucleic acid was made soluble in 5% trichloroacetic acid. The reaction was stopped by adding 0.1 M EDTAto make a final concentration of 5 mM and cooling the reaction mixture in an ice bath. The reaction mixture was dialysed against 10 mM Tris-HCl buffer, pH 7.8, containing 0.2 mM EDTA (Tris-EDTA buffer). The nuclear debris and the insoluble particulate material were removed by centrifuging the mixture at 12,000 x g for 30 riin. The clear supernatant contained nucleosomes and oligonucleosomes. Fractionation of Nucleosomes The nucleosome preparation(20-30 A260in 2 ml)was applied toa Bio-Gel A-5m column(llOcm x 1 .6cm)previously equilibrated with Tris-EDTA buffer (29). Flow rate was maintained at 5ml/cm2/hr and 2-mlfractions were collected.Nucleosomes and oligonucleosomes were obtained bypooling appropriatefractionsfrom the column. 854
Nucleic Acids Research Turbidity Measutements
Five microliters of either 2 M(NH4)2S04 or 2 M KC1 were added successively to the nucleosomal solution in Tris-EDTA buffer and the absorbance at 420 nm was monitored after each addtion. Turbidity was also measured in the presence of 10 mMM MgCl2. Circular Dichroism Measurements The circular dichroism spectra were measured on a Durrum Jasco CD-SP spectrophotometer at room temperature. The nucleosome concentration in each sample was such that the equivalent DNA concentration was always in the range of 50-80 ig/ml (similar to the one usedin chromatin transcription reactions) (30-32). The samples were prepared by mixing appropriate amounts of 1 M MgCl 29 2 M (NH4)2S04, or 2 M KC1 and stock solutions of nucleosomes and diluting the mixture with Tris-EDTA buffer. The value of {t}, the molar ellipticity, was calculated at 2-nm intervals, and plotted against wavelength. The DNA concentration in the nucleosomeswas determined using E260=6600' (3,6).
Polyacrylamide Gel Electrophoresis Three percent composite cylindrical gels(2.5% acrylamide(19:1, acrylamide: bis-acrylamide) 0.5% agarose)were employed to analyze the pooled nucleosomes from the Bio-Gelcolumn.The length of DNAfrom nucleosomes or oligonucleosomes was determined on 5.5% composite cylindrical gels(5%acrylamide (19: 1, acrylamide: bis-acrylamide) and 0.5% agarose) (10). Restriction fragments of PM2 DNA generated by restriction enzyme Hae III were run on a parallel gel as markers. Bromophenol blue was used to indicate the extent of migration on the gel. The running buffer was 40 mM Tris-HCl, pH 7.8, 20 mM sodium acetate, and 3 mM EDTA. The gels were run at a constant current of 2 mA per tube. The gels were stained in 2 lag/ml ethidium bromide solution in running buffer for 30 min and scanned on a Aminco Bowmen fluorospectrophotometer.The exciting wavelength was 510 nm and the fluorescence was measured at 590 nm.
Isolation of DNA The DNA from nucleosomes and oligonucleosomes was purified by phenol extraction (33). Phenol was redistilled and saturated with 0.01 M Tris-HCl, pH 7.8, containing 0.2 M NaCl and 0.2% 8-hydroxyquinoline, before use.
RESULTS Nucleosomes and Oligonucleosomes Nucleosomes and oligonucleosomes prepared by digestion of human placental 855
Nucleic Acids Research nuclei with staphylococcal nuclease were fractionated by gel filtration (Bio-Gel A-5m) chromatography. The elution profile (Fig. 1) can be resolved into four Gaussian curves. Pool I (fractions 63-68) contains more than 90% mononucleosomes with less than 10% contamination of dinucleosomes. If only the trailing side of the peak is used(fractions 66-70),there is no dinucleosome contamination. When the material from pool I is analyzed by electrophoresis on a 3% composite gel, a peak of nucleosomes and a small contaminating peak of dinucleosomes (Fig. 2) is observed. On the other hand, the material in pool II (fractions 40-58) contains mainly dinucleosomes and trinucleosomes with smaller amounts of mononucleosomes and tetranucleosomes (Fig. 2). Nucleosomes were analyzed on SDS-urea polyacrylamide(12.5%)gels and nucleosomal proteins stained with Coomasie blue.These gels show the presence of all 4histones invo'vedin the nucleosomal core as well as histone Hl. The ratio of the amount of HI to the amount of H4 suggests that >70% of the nucleosomes do not have Hl. The DNAs extracted from pools I and II were run on 5.5% composite cylindrical gels (Fig. 3). The repeat length of DNAis calculated tobe 185base pairs and the length of DNA
Gel Filtration (A-5m) Column of Nuclear Digest.
1.4 ~~~~l0~~~~~ E
°) 1.0
-
Pdl
t
OIimh_deommss
_-I
C>O.6
0.2 40
60 Fraction Number
80
Purified nuclei from human placenta of staphylococcal nuclease. The nuclear anAuWe clear supernatant fractionated on a Bio-
Figure 1. Gel filtration of the nuclear digest. were digested for 12 min with 10 units/A debris was removed by centrifugation
Gel A-5m column.
856
Nucleic Acids Research involved in the core is about 155 base pairs. Both these values compare well with the values for nucleosomal and core DNAs obtained from various tissues (10,1 1) . Turbidity Measurements The amount of insoluble or aggregated macromolecules in a suspension can be estimated from the absorbance at 420 nm. We have used this property to determine the effectsof salt concentrationon the solubilityof nucleosomesand oligonucleosomes. The turbidity measurements done on the nucleosome solutions indicate that the nucleosomes are partially insoluble over a wide range of {(NH4)2S04}(Fig. 4).There is a peak of turbidity at about 50 mM (NH4)2S04 for nucleosomes, while a broad peak in the range of 70-120 mM (NH4)2S04is observed for oligonucleosomes. Similarly, chromatin is partially insoluble over a broad range of NaCl concentrations (34). In order to examine the nature of this insolubility, we measured the effect of nucleosome concentration on turbidity (Fig. 5) in 50 mM (NH4)2S04. The linear relationship between nucleosome concentration and insolubility suggests that the state of aggregation of nucleosomes remains the same in the range of nucleosome concentration studied here.
Gel Electrophoresis of Nucleosomes and Oligonucleosomes. 2.5 % Acry lamide - 0.5 %/o Agarose Mono-
Pool I
Bottom
|
i -nuc le osomes ~~~~~Tr
Top
Figure 2. Analysis of nucleosomes by polyacrylamide gel electrophoresis. Pool I (fractions 63-68) and pool 11 (fractions 40-58) were run on 3. 0%6 composite cylindrical gels. The gels were stained with 2 ,g/ml ethidium bromide solution in running buffer and scanned with a fluorospectrophotometer. Fluorescence intensity is plotted versus migration distance.
857
Nucleic Acids Research Gel Electrophoresis of DNA from Nucleosomes and Oligonucleosomes (5% Acrylamide-0.5%Agarose) A.PM-2 DNA
HoellIdigest
592
288 ~n642
160
B. Pool I DNA
l5gl\-l~~~~~85
o
155
i
_LL
C. Pool IIDNA
410 Trimer
Monomer
Bottom
-
Migration
1610
Top
Figure 3. Length of nucleosomal DNA. Ihe DNA purified by phenol extraction of material from pools I and II was electrophoresed on 5. 5% composite cylindrical gels. The fluorescent scans were obtained after staining the gels with ethidium bromide. (A) PM-2 DNA fragments generated with Hae III restriction enzyme, used as markers. (B) DNA from material in pool I. (C) DNA from material in pool II. Migration was from right to left.
Effect of Salt on Turbidity of Nucleosomes and Oligonucleosomes
I cw
I @0N
-2
m
,0
[(NH4k2 S04]in mM Figure 4. Effect of (NH4) SO on the turbidity of nucleosomes and oligonucleosomes. To a nucleosome solution 60-A0 Mg) in Tris buffer 5 , I aliquots of 2 M (NH4) S04 solution Were added and mixed; the absorbance at 420 nm was read after each addition. ( o -o -) nucleosome (pool I) at a concentration of 60 ,Ag. (- *- oligonucleosomes (pool II) at a concentration of 70 Mg/ml. 858
)
Nucleic Acids Research Effect of Nucleosome Concentration on Turbidity in 0.05 M [(NH4)2 S04]
0.14 0.12 E
0.10
¢ 0.08 -
0.06
