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Affinity chromatography of chromatin on single-stranded DNA-agarose columns
ANALYTICAL
BIOCHEMISTRY
Affinity
107, 1?4- 127 (1980)
Chromatography of Chromatin on Single-Stranded DNA-Agarose Columns PETER
NEHLS’
AND
Received
MANFRED
April
RENT
38. 1980
Passing chromatin fragments of rat liver nuclei through DNA-agarose columns results in the removal of all histones of the HI clans and almost all (~95%~) nonhistone proteins from the chromatin and thus leads to the separation of DNA molecules containing nucleosomal histones only. Elution of the porteins bound to DNA-agarose by salt gradients leads to a fractionation of chromosomal proteins indicating that they bind with different affinities to single-stranded DNA. This simple and fast procedure i\ suitable for both the isolation of histone HI-depleted chromatin and the fractionation of nonhistone proteins.
Many of the nonhistone proteins are thought to be involved in gene regulation by binding to specific DNA sequences ( 1.2). In procaryotes it has been shown that in addition to the specific repressor-operator interaction weak unspecific binding of repressor molecules to nucleic acids occurs (3). It seems therefore interesting to ask how strongly chromosomal proteins interact with nucleic acids in general until assays for eucaryotic regulator proteins are available. For our studies we exploited the finding that some of the chromosomal proteins are transferred from chromatin to exogenous nucleic acids (4.5). We modified this procedure by immobilizing DNA in agarose (6) which was then used for competition experiments.
with EDTA (final concentration 5 mM). Solubilized chromatin fragments (more than 70% of total chromatin) were separated from the residual nuclear pellet by centrifugation (8000,~ , 2 min) and gel-chromatographed on a Sepharose 4B column ( 1.5 cm’ K 20 cm) with a solution containing 100 rn,u NaCI. 5 rnM Tris-HC1 (pH 7.5), and 0.1 mM EDTA. For solubility experiments chromatin fragments (I mgiml) were dialyzed against buffers containing different concentrations of NaCI. The amount of soluble chromatin was determined after centrifugation at 8000,~ for 4 min. Affinity chromatography of chromatin fragments was performed on single-stranded DNAagarose columns (6). Columns (5 mg calf thymus DNA/ml. 3 cm” Y 7 cm) were loaded with 6 ml of a solution containing 8 mg chromatin and 100 rnhl NaCI buffer at a pressure of 20 cm and finally eluted with buffers containing different concentrations of NaCl.
EXPERIMENTAL PROCEDURES Rat liver nuclei (8) have been digested with micrococcal nuclease (3.3 x 10Hnuclei with 60 units in a volume of I ml) in the presence of 60 mM NaCl. I mM MgCI,. I mM CaCI,, and 5 mM Tris-HCI (pH 7.5) at 37°C for 15 s. The reaction was stopped
((1) Sc~llrhilif~ c!f’ Cl~r~~i~zr~fir~
’ Prewnt addre\\: Institut fur Zellbiologie. forschung. Univerktt Essen, Hufeland\tr. Es\en I. Federal Republic of Germany.
Chromatin fragments were isolated in the presence of 60 rnbt NaCl and dialyzed
Tumor55. 4300
RESULTS
AND DISCUSSION
AFFINITY
m
NOCI
CHROMATOGRAPHY
to chromatin fragments. The transferred proteins can be released from the column with 2 M NaCl (Fig. 3~). They consist of nonhistone proteins. histones of the Hl class, and a very small amount of nucleosomal histones.
[mMl
FIG. I. Solubility of rat liver chromatin at different concentrations of NaCl.
fragments
against buffers containing different concentrations of NaCl. Chromatin forms a precipitate in solutions containing NaCl between about 140 and 400 mM (Fig. 1). At both lower and higher salt concentrations chromatin fragments are soluble (Fig. 1) and can therefore be used for chromatographic purposes.
Nonbound nuclear proteins were separated from chromatin by gel filtration in 100 mM NaCl. Chromatin fragments eluted with the column void volume; they were used for competition experiments with immobilized DNA by allowing them to penetrate into a DNA-agarose column which was then washed with 100 mM NaCl buffer. The result of a typical experiment is shown in Fig. 2. More than 90% of the chromosomal DNA (A2,i,,) eluted with the void volume of the column. The small amount of chromosomal DNA retained by the column can be released with 2 M NaCl (Fig. 2). Protein components of different fractions have been analyzed by SDS’-gel electrophoresis (Fig. 3). Figure 3a shows the protein pattern of rat liver chromatin which was used for DNA-agarose affinity chromatography. Figure 3b represents proteins of the material which elutes with the void volume. The difference is striking. Almost all chromosomal proteins but the nucleosomal histones must have been transferred to the column since they are no longer bound ” Abbreviation
used:
SDS,
sodium
125
OF CHROMATIN
dodecyl
sulfate.
( c) Ftxctiotlcttiott
of’ Cltrotttosottltrl
Most of the chromosomal proteins which were transferred to DNA-agarose columns interact with the single-stranded DNA of the column with different affinities. This can be shown by eluting the column with solutions containing different concentrations of NaCl (Fig. 4). Many proteins are released already with 0.2 M NaCl (Fig. 4~). They consist mainly of large proteins (>4 x IO” (d)) and a small amount of nucleosomal histones. The protein pattern which results from the elution with 0.4 M NaCl is different (Fig. 4d). There are four prominent protein bands. At 0.7 M NaCl histones ofthe HI class are eluted almost exclusively (Fig. 4e). Elution with 2 M NaCl releases tightly bound proteins which are small in number and amount (Fig. 4f ); they include also nucleosomal histones. More than a decade ago it was found that some chromosomal proteins can be transferred to exogenous nuclei acids (4). Systematic studies revealed that histones of
2(
2M Nacl
0 x 4 10
FIG. 2. Chromatography of chramatin on calf thymus DNA-agarose columns. Proteins bound to columns were released with 2 M NaCl buffer at the \ame pressure
126
NEHLS
AND
the HI class can be removed specifically from isolated chromatin when the exogenous nucleic acid was used in vast excess (about 20 times as much as chromatin) (5). In those experiments chromatin and exogenous nucleic acids must differ in fragment size in order to separate the two components physically after protein exchange took place. To simplify this procedure we immobilized the exogenous nucleic acid (6). By passing chromatin fragments through DNA-agarose columns similar exchange reactions occur as in solution: All histone H 1 molecules are transferred to the column DNA (Fig. 3b,c) when the amount of DNA in the column exceeds 10 to 30 times the chromosomal DNA. At the same time almost all of the mass of nonhistone proteins (295%) are transferred to the column as well (Fig. 3b,c). With our method histone HI- and nonhistone-depleted chromatin fragments (unfolded nucleosome chains) can be produced
RENZ
a FIG. proteins.
b
e
4. Analysis DNA-agarose
liver
chromatin
NaCl and
buffer later
centrations and (f) marker
cd of
fractionated columns were
fragments and with of
2.0 proteins
M
eluted buffers NaCI: NaCl.
(a) first
dissolved
with the containing
(c) 0.2 M, td) The far right
of different
marker
f
molecular
chromosomal loaded with
rat
in
mM
100
same buffer (b) different con0.4 M. (e) 0.7 panel represents
M.
weight\.
in a simple way without exposing them to low ionic strength. At the same time nonhistone proteins can be fractionated according to their affinities to single-stranded DNA. After completion of this work, Allan ct (11. (9) reported that histone HI-depleted chromatin fragments can be produced by recycling chromatin through a DNA-cellulose column. Recycling is not needed when DNA-agarose columns are used probably due to their lower flow rate. a FIG. 3. SDS-polyacrylamide 17) of proteins of rat liver
b
c gel chromatin
REFERENCES electrophoresis fragments
fore (a) and after (b) chromatography agarose columns. The protein pattern after column with 2 M NaCl is shown in tc).
be-
on DNAeluting the
I.
Yamamoto. K. R., and Albert>, B. M. Atlnrr. I?<>\,. Rir~.k~m. 45, 721-746. 2. Gierer. A. t 1974) Cold Spring Hnrhr~ c)uunf. Bird. 38. 95 I-96 I.
(1976) .Sv~zp.
AFFINITY
3. Lin.
S.. and
Riggs.
A.
CHROMATOGRAPHY
D. (1972)
J. ‘MoI.
Bicjl.
OF
72.
Kurz.
4. Chalkley. R.. and Jewen, ~h~vrli.\try 7, 4388-4395. 5.
Ilyin, U.
Y. N..
V.. Varshavsky. and Georgiev.
Bicx.Ifcrtr. 6.
Schaller.
Ch..
and
Bioc~Ilem.
671-690. R.
H.
(1968)
Bio-
7.
Ntihslein.
Laemmli.
Nictrwhmann, I.
I. (I9771
Euf.
(1970)
I Lc~rftlc~rr)
./.
26, 474-48 U.
K.
Nlrturc
227.
680-685. A. Ya.. Mickelbaar-, G. P. ( 1971) Eur.
8. 9.
Ch..
Bonhoeffrr.
F.
J..
Blobel.
G..
and
Potter.
R. V. I 1966)
.Scic~~(~~, 154,
1667.
,I.
22, 235-345. H..
137
CHROMATIN
Allan.
Prcw.
.I..
Staynov.
D.
Z..
and
,Vrir. .4(.C/d. .S(,i. o.s.1.
Gould,
H.
77. 882-889.
t 1980)
BIOCHEMISTRY
Affinity
107, 1?4- 127 (1980)
Chromatography of Chromatin on Single-Stranded DNA-Agarose Columns PETER
NEHLS’
AND
Received
MANFRED
April
RENT
38. 1980
Passing chromatin fragments of rat liver nuclei through DNA-agarose columns results in the removal of all histones of the HI clans and almost all (~95%~) nonhistone proteins from the chromatin and thus leads to the separation of DNA molecules containing nucleosomal histones only. Elution of the porteins bound to DNA-agarose by salt gradients leads to a fractionation of chromosomal proteins indicating that they bind with different affinities to single-stranded DNA. This simple and fast procedure i\ suitable for both the isolation of histone HI-depleted chromatin and the fractionation of nonhistone proteins.
Many of the nonhistone proteins are thought to be involved in gene regulation by binding to specific DNA sequences ( 1.2). In procaryotes it has been shown that in addition to the specific repressor-operator interaction weak unspecific binding of repressor molecules to nucleic acids occurs (3). It seems therefore interesting to ask how strongly chromosomal proteins interact with nucleic acids in general until assays for eucaryotic regulator proteins are available. For our studies we exploited the finding that some of the chromosomal proteins are transferred from chromatin to exogenous nucleic acids (4.5). We modified this procedure by immobilizing DNA in agarose (6) which was then used for competition experiments.
with EDTA (final concentration 5 mM). Solubilized chromatin fragments (more than 70% of total chromatin) were separated from the residual nuclear pellet by centrifugation (8000,~ , 2 min) and gel-chromatographed on a Sepharose 4B column ( 1.5 cm’ K 20 cm) with a solution containing 100 rn,u NaCI. 5 rnM Tris-HC1 (pH 7.5), and 0.1 mM EDTA. For solubility experiments chromatin fragments (I mgiml) were dialyzed against buffers containing different concentrations of NaCI. The amount of soluble chromatin was determined after centrifugation at 8000,~ for 4 min. Affinity chromatography of chromatin fragments was performed on single-stranded DNAagarose columns (6). Columns (5 mg calf thymus DNA/ml. 3 cm” Y 7 cm) were loaded with 6 ml of a solution containing 8 mg chromatin and 100 rnhl NaCI buffer at a pressure of 20 cm and finally eluted with buffers containing different concentrations of NaCl.
EXPERIMENTAL PROCEDURES Rat liver nuclei (8) have been digested with micrococcal nuclease (3.3 x 10Hnuclei with 60 units in a volume of I ml) in the presence of 60 mM NaCl. I mM MgCI,. I mM CaCI,, and 5 mM Tris-HCI (pH 7.5) at 37°C for 15 s. The reaction was stopped
((1) Sc~llrhilif~ c!f’ Cl~r~~i~zr~fir~
’ Prewnt addre\\: Institut fur Zellbiologie. forschung. Univerktt Essen, Hufeland\tr. Es\en I. Federal Republic of Germany.
Chromatin fragments were isolated in the presence of 60 rnbt NaCl and dialyzed
Tumor55. 4300
RESULTS
AND DISCUSSION
AFFINITY
m
NOCI
CHROMATOGRAPHY
to chromatin fragments. The transferred proteins can be released from the column with 2 M NaCl (Fig. 3~). They consist of nonhistone proteins. histones of the Hl class, and a very small amount of nucleosomal histones.
[mMl
FIG. I. Solubility of rat liver chromatin at different concentrations of NaCl.
fragments
against buffers containing different concentrations of NaCl. Chromatin forms a precipitate in solutions containing NaCl between about 140 and 400 mM (Fig. 1). At both lower and higher salt concentrations chromatin fragments are soluble (Fig. 1) and can therefore be used for chromatographic purposes.
Nonbound nuclear proteins were separated from chromatin by gel filtration in 100 mM NaCl. Chromatin fragments eluted with the column void volume; they were used for competition experiments with immobilized DNA by allowing them to penetrate into a DNA-agarose column which was then washed with 100 mM NaCl buffer. The result of a typical experiment is shown in Fig. 2. More than 90% of the chromosomal DNA (A2,i,,) eluted with the void volume of the column. The small amount of chromosomal DNA retained by the column can be released with 2 M NaCl (Fig. 2). Protein components of different fractions have been analyzed by SDS’-gel electrophoresis (Fig. 3). Figure 3a shows the protein pattern of rat liver chromatin which was used for DNA-agarose affinity chromatography. Figure 3b represents proteins of the material which elutes with the void volume. The difference is striking. Almost all chromosomal proteins but the nucleosomal histones must have been transferred to the column since they are no longer bound ” Abbreviation
used:
SDS,
sodium
125
OF CHROMATIN
dodecyl
sulfate.
( c) Ftxctiotlcttiott
of’ Cltrotttosottltrl
Most of the chromosomal proteins which were transferred to DNA-agarose columns interact with the single-stranded DNA of the column with different affinities. This can be shown by eluting the column with solutions containing different concentrations of NaCl (Fig. 4). Many proteins are released already with 0.2 M NaCl (Fig. 4~). They consist mainly of large proteins (>4 x IO” (d)) and a small amount of nucleosomal histones. The protein pattern which results from the elution with 0.4 M NaCl is different (Fig. 4d). There are four prominent protein bands. At 0.7 M NaCl histones ofthe HI class are eluted almost exclusively (Fig. 4e). Elution with 2 M NaCl releases tightly bound proteins which are small in number and amount (Fig. 4f ); they include also nucleosomal histones. More than a decade ago it was found that some chromosomal proteins can be transferred to exogenous nuclei acids (4). Systematic studies revealed that histones of
2(
2M Nacl
0 x 4 10
FIG. 2. Chromatography of chramatin on calf thymus DNA-agarose columns. Proteins bound to columns were released with 2 M NaCl buffer at the \ame pressure
126
NEHLS
AND
the HI class can be removed specifically from isolated chromatin when the exogenous nucleic acid was used in vast excess (about 20 times as much as chromatin) (5). In those experiments chromatin and exogenous nucleic acids must differ in fragment size in order to separate the two components physically after protein exchange took place. To simplify this procedure we immobilized the exogenous nucleic acid (6). By passing chromatin fragments through DNA-agarose columns similar exchange reactions occur as in solution: All histone H 1 molecules are transferred to the column DNA (Fig. 3b,c) when the amount of DNA in the column exceeds 10 to 30 times the chromosomal DNA. At the same time almost all of the mass of nonhistone proteins (295%) are transferred to the column as well (Fig. 3b,c). With our method histone HI- and nonhistone-depleted chromatin fragments (unfolded nucleosome chains) can be produced
RENZ
a FIG. proteins.
b
e
4. Analysis DNA-agarose
liver
chromatin
NaCl and
buffer later
centrations and (f) marker
cd of
fractionated columns were
fragments and with of
2.0 proteins
M
eluted buffers NaCI: NaCl.
(a) first
dissolved
with the containing
(c) 0.2 M, td) The far right
of different
marker
f
molecular
chromosomal loaded with
rat
in
mM
100
same buffer (b) different con0.4 M. (e) 0.7 panel represents
M.
weight\.
in a simple way without exposing them to low ionic strength. At the same time nonhistone proteins can be fractionated according to their affinities to single-stranded DNA. After completion of this work, Allan ct (11. (9) reported that histone HI-depleted chromatin fragments can be produced by recycling chromatin through a DNA-cellulose column. Recycling is not needed when DNA-agarose columns are used probably due to their lower flow rate. a FIG. 3. SDS-polyacrylamide 17) of proteins of rat liver
b
c gel chromatin
REFERENCES electrophoresis fragments
fore (a) and after (b) chromatography agarose columns. The protein pattern after column with 2 M NaCl is shown in tc).
be-
on DNAeluting the
I.
Yamamoto. K. R., and Albert>, B. M. Atlnrr. I?<>\,. Rir~.k~m. 45, 721-746. 2. Gierer. A. t 1974) Cold Spring Hnrhr~ c)uunf. Bird. 38. 95 I-96 I.
(1976) .Sv~zp.
AFFINITY
3. Lin.
S.. and
Riggs.
A.
CHROMATOGRAPHY
D. (1972)
J. ‘MoI.
Bicjl.
OF
72.
Kurz.
4. Chalkley. R.. and Jewen, ~h~vrli.\try 7, 4388-4395. 5.
Ilyin, U.
Y. N..
V.. Varshavsky. and Georgiev.
Bicx.Ifcrtr. 6.
Schaller.
Ch..
and
Bioc~Ilem.
671-690. R.
H.
(1968)
Bio-
7.
Ntihslein.
Laemmli.
Nictrwhmann, I.
I. (I9771
Euf.
(1970)
I Lc~rftlc~rr)
./.
26, 474-48 U.
K.
Nlrturc
227.
680-685. A. Ya.. Mickelbaar-, G. P. ( 1971) Eur.
8. 9.
Ch..
Bonhoeffrr.
F.
J..
Blobel.
G..
and
Potter.
R. V. I 1966)
.Scic~~(~~, 154,
1667.
,I.
22, 235-345. H..
137
CHROMATIN
Allan.
Prcw.
.I..
Staynov.
D.
Z..
and
,Vrir. .4(.C/d. .S(,i. o.s.1.
Gould,
H.
77. 882-889.
t 1980)