|
|
|
|||||||
|
|||||||||
(Received for publication, August 28,
1995; and in revised form, September 22, 1995) From the
We find that reconstituted nucleosome cores containing specific
DNA sequences dissociate on dilution. This disruption of histone-DNA
contacts leading to the release of free DNA is facilitated by the
presence of the core histone tails, MgCl
The nucleosome core is a fragile object (van Holde, 1988).
Systematic study of the stability of nucleosome cores isolated from the
nuclei of somatic cells has determined the temperature, pH, and salt
concentrations at which histone-DNA interactions are disrupted (Zama et al., 1978; Gordon et al., 1979; Libertini and
Small, 1980; Burch and Martinson, 1980; Burton et al., 1978;
Walker and Wolffe, 1984). An important aspect of nucleosome core
stability is the sensitivity of histone-DNA interactions to dilution.
The fraction of intact nucleosome cores decreases as the total
nucleosome concentration is lowered (Stacks and Schumaker, 1979; Lilley et al., 1979; Cotton and Hamkalo, 1981; Eisenberg and
Felsenfeld, 1981; Yager and van Holde, 1984; Ausio et al.,
1984a, 1984b). This dissociation of nucleosome cores into histones and
free DNA under dilute conditions is substantial at physiological ionic
strengths. Cotton and Hamkalo(1981) found that more than 20% of the
nucleosome cores would dissociate at a concentration of 10 ng/µl at
physiological ionic strength over a 2-h period. Lilley et
al.(1979) in determining the consequences for nucleosome integrity
of association with eukaryotic RNA polymerase II found that ``at
the low nucleosome concentrations used to achieve enzyme excess for
nucleosome transcription experiments, dissociation to free DNA is
considerable, irrespective of the presence of polymerase.'' More recent work has made extensive application of nucleosome cores
reconstituted using defined sequences of DNA (Archer et al.,
1991; Chen et al., 1994;
Côtéet al., 1994;
Hayes and Wolffe, 1992; Imbalzano et al., 1994; Kwon et
al., 1994; Lee et al., 1993; Li et al., 1994; Li
and Wrange, 1993; Perlmann and Wrange, 1988; Pina et al.,
1990; Workman and Kingston, 1992). Radiolabeling of the DNA used in
these experiments has facilitated the use of very dilute solutions. In
certain instances nucleosome disruption directed by trans-acting
factors has been documented (Chen et al., 1994;
Côtéet al., 1994;
Imbalzano et al., 1994; Kwon et al., 1994; Workman
and Kingston, 1992). Most of these experiments make use of
reconstituted nucleosomes under dilute conditions ranging from 6
ng/µl (Workman and Kingston, 1992) to 0.1 ng/µl (Imbalzano et al., 1994). Thus it is possible that spontaneous nucleosome
disruption might influence the outcome of these experiments. We have
examined the integrity of nucleosome cores reconstituted so as to
contain the Xenopus borealis 5 S RNA gene. DNA sequences of
this type are among those with the highest affinity for the histone
octamer (Shrader and Crothers, 1989; Schild et al., 1993). The X. borealis 5 S RNA gene also directs the positioning of the
histone octamer with respect to DNA sequence, offering the opportunity
to examine the consequences of nucleosome dissociation on the DNase I
cleavage of DNA within a positioned nucleosome core (Rhodes, 1985;
Hayes et al., 1990). We find that the 5 S nucleosome and other
reconstituted nucleosomes dissociate on dilution under the standard
binding conditions for transcription factors such as TBP.
Figure 5:
Disruption is not dependent on DNA
sequence or specific to monomeric core particles. Nucleosomes
reconstituted onto the indicated DNA fragments and incubated under
standard conditions (see ``Materials and Methods''). Lanes 1-5 contain nucleosome core particles, and lanes 6-10 containtwo core particles reconstituted onto
a longer fragment of DNA. Histone-bound and free DNA are
indicated.
Figure 2:
DNase I cleavage of naked DNA and
nucleosomal DNA isolated from native gels and of disrupted nucleosomes
in solution. A, sucrose gradient-purified core particles were
diluted to 1 ng/µl before digestion with DNase I, and nucleoprotein
complexes were then resolved on a non-denaturing gel before elution,
deproteinization, and resolution on a denaturing gel. The digestion
pattern of ``free DNA'' (lane 1) and nucleosomal DNA (lane 2) is shown. Arrowheads indicate the
10-11-bp pattern of DNase I cleavage in nucleosomal DNA. B, sucrose gradient-purified core particles at the indicated
concentrations (per µl) after cleavage with DNase I. MgCl
Figure 1:
Nucleosome
disruption and TBP binding. Nucleosome core particles at the indicated
concentrations (per µl) were incubated in the presence and absence
of TBP and TFIIA. Lanes 1 and 2 contain naked DNA; lanes 3-8, core particles; and lanes 9 and 10, core particles from which the histone tails have been
removed by trypsin (TrypOct) (see ``Materials and
Methods''). Lanes 2, 6, 7, 8, and 10 are with
the addition of TBP and TFIIA to 1
We next examined the binding of TBP and
TFIIA to free and nucleosomal DNA. The TBP/TFIIA proteins bind to the
TATA box within naked DNA but not to the TATA box when it is associated
with an unmodified octamer of histones (Fig. 1, lanes
6-8). Incubation of nucleosomes with TBP/TFIIA under
progressively more dilute conditions leads to a progressively larger
proportion of TBP/TFIIA bound to naked DNA appearing with dilution (Fig. 1, lanes 6-8,Table 1). This is
because histone-DNA contacts are selectively disrupted on dilution.
Thus a potential contributory factor in the access of TBP to
nucleosomal DNA (Imbalzano et al., 1994) could be the
dissociation of nucleosomes at high dilution. Imbalzano et
al.(1994) use reconstituted nucleosomes at a very low
concentration of 0.1 ng/µl. As a control for the capacity of our
gel system to resolve a tertiary complex of histones, TBP/TFIIA, and
DNA, we examined the binding of TBP/TFIIA to nucleosomal particles from
which the core histone tails had been removed using trypsin
(``tail-less nucleosomes''). Earlier work had indicated that
the major impediment to TFIIIA access to DNA in a nucleosome containing
the X. borealis 5 S rRNA gene was the interaction of the core
histone tails with DNA (Lee et al., 1993). TBP/TFIIA
efficiently forms a tertiary complex with the tail-less nucleosome
containing the TATA box (Fig. 1, lanes 9 and 10). Thus the core histone tails impede TBP/TFIIA access to
nucleosomal DNA under these conditions. A concern in this type of
analysis is that gel electrophoresis might introduce a potential
artifact. Nucleosomes might dissociate upon entry into the gel. An
experiment that might verify or eliminate any gel dissociation artifact
is to carry out a DNase I footprinting cleavage, followed immediately
by separation into free DNA and nucleosomes on a non-denaturing gel
(Wolffe and Hayes, 1993). The different bands can then be assayed for
cleavage pattern after elution and deproteinization in a denaturing
gel. If the ``free DNA'' is really free, then it should show
no 10-11-bp periodicity in cleavage. Such a 10-11-bp
periodicity in cleavage might reflect a nucleosomal organization in
solution that is lost on electrophoresis. We carried out this
experiment using nucleosomal DNA in dilute solution (1 ng/µl) and
found that ``free DNA'' in the gel was cleaved by DNase I
without any 10-11-bp periodicity, i.e. it is digested as
naked DNA (Fig. 2A, lane 1). In contrast,
nucleosomal DNA isolated from the native gel showed a clear
10-11-bp periodicity of cleavage (Fig. 2A, lane 2). This result suggests that nucleosomes do not
dissociate during electrophoresis under our experimental conditions. It
should also be noted that there is little smearing of nucleosomal DNA,
reflecting the continued stability of the complex once it has entered
the gel matrix. A feature of nucleosomal disruption by the yeast or
human SWI/SNF complexes is the loss of DNase I cleavage patterns
characteristic of the nucleosome
(Côtéet al., 1994;
Imbalzano et al., 1994). Consistent with earlier data on mixed
sequence nucleosomes (Ausio et al., 1984a, 1984b) and the 5 S
nucleosome containing a TATA box (Fig. 1), dilution of
nucleosomes will contribute to their disruption. In fact, DNase I
cleavage of reconstituted nucleosomes incubated under progressively
more dilute conditions leads to the progressive loss of protection from
DNase I cleavage (Fig. 2B, lanes 2-4).
In the earlier published experiments
(Côtéet al., 1994;
Imbalzano et al., 1994) nucleosomes are not isolated from
native gels following DNase I cleavage (Wolffe and Hayes, 1993); thus
mixed populations of free DNA and histone-bound DNA might complicate
interpretation of the experimental results. Moreover, the efficiency of
reconstitution into nucleosomes might vary with DNA template; for
example Shrader and Crothers(1989) report wide variation in the
stability of DNA-histone interactions. Without examining nucleoprotein
complexes on native gels or by analytical ultracentrifugation it is
almost impossible to determine reconstitution efficiencies. We
suggest that it is important to control for nucleosome dissociation
under the dilution conditions used in in vitro experiments
when assessing trans-acting factor access to nucleosomal DNA or the
disruption of nucleosomes by molecular complexes such as SWI/SNF.
Figure 3:
Effect of histone tails on nucleosome
disruption. A, the stability of nucleosome core particles with
histone tails removed by trypsin compared with non-treated core
particles upon dilution. MgCl
Figure 4:
Effect of MgCl
Nucleosome cores containing the
histone tails are less stable to dilution than cores from which the
histone tails have been removed by trypsin (Fig. 3A).
The tail-less octamers remain stable if MgCl
Our next
experiments examined the role of temperature and KCl concentration in
nucleosome dissociation. In agreement with earlier work (Ausio et
al., 1984b) we find that an increase in temperature to 37 °C
and monovalent cation concentration (KCl) to 280 mM further
destabilizes nucleosome cores (Fig. 4, B and C). An increase in divalent or monovalent cation
concentrations will contribute to the release of the core histone tails
from stable interaction with nucleosomal DNA (Walker, 1984). This
release might facilitate nucleosome disruption, potentially by allowing
the tails to make contacts outside of the nucleosome. In these studies
we have also found an example of two octamers bound to a single DNA
fragment (Fig. 4C, lanes 1, 5, and 9) as previously reported (Ausio et al., 1984a,
1984b). The upper complex is selectively destabilized by dilution,
reflecting the weaker association of the second histone-octamer with
DNA (Ausio et al., 1984b). Finally we made use of two
additional specific chromatin substrates to show that a mononucleosome
containing sequences from the Xenopus TR
Volume 270,
Number 46,
Issue of November 17, 1995 pp. 27399-27402
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
THE EFFECT OF PARTICLE CONCENTRATION, MgCl
AND KCl
CONCENTRATION, THE HISTONE TAILS, AND TEMPERATURE (*)
(5 mM),
KCl (60 mM), and temperatures above 0 °C. Under reaction
conditions that are commonly used to assess trans-acting factor access
to nucleosomal DNA, histone-DNA contacts are on the threshold of
instability. We demonstrate how dilution of reconstituted nucleosomes
containing a TATA box can facilitate TBP access to DNA.
DNA Constructs
Unless otherwise noted, all
experiments were performed with a modified Xenopus 5 S RNA
gene in which the adenovirus major late promoter TATA box (TATAAAAG)
replaces native sequence from -73 to -66 relative to the
start site of 5 S RNA gene transcription (+13; see Pruss et
al.(1995)). This replacement was obtained by polymerase chain
reaction mutagenesis of plasmid XP-10 (Wolffe et al., 1986).
The construct was digested with EcoRI and RsaI to
liberate a 154-bp (
)fragment. For Fig. 5the
following constructs were used: a 159-bp DNA fragment that was
amplified using polymerase chain reaction from the Xenopus TR
A gene (from +322 to +163 relative to the start
site of transcription at +1 (Ranjan et al., 1994)) and a
424-bp DNA fragment obtained by digestion of a plasmid containing two
tandem repeats of the X. borealis somatic 5 S RNA gene
(pX5S197-2) (Ura et al., 1995). All DNA fragments were
end-labeled with T4 polynucleotide kinase or Klenow fragment (New
England BioLabs) and gel purified.
Transcription Factors
Recombinant Saccharomyces cerevisiae TBP (Hoffman and Roeder, 1993;
Horikoshi et al., 1989) was expressed in and purified from Escherichia coli as described (Nikolov et al., 1992)
except that trypsin cleavage of the histidine-tag was omitted.
Recombinant S. cerevisiae TFIIA was a kind gift of Dr.
Yoshihiro Nakatani at NIH.Nucleosome Reconstitution
Nucleosome core
particles were purified from chicken erythrocyte nuclei as described
previously (Wolffe and Hayes, 1993). Trypsinized octamers were prepared
as described by Wolffe and Hayes(1993).Dilution Conditions
Unless noted, standard buffer
conditions for TBP/TFIIA binding to DNA were used throughout.
Reconstituted nucleosome core particles were diluted to the desired
concentrations in TE (10 mM Tris, pH 8.0, 1 mM EDTA)
in a volume of 7.2 µl before being mixed with the following buffer
in 25-µl reactions. The final buffer concentration after mixing was
20 mM HEPES/KOH, pH 7.8, 5.6 mM dithiothreitol, 12
mM Tris-HCl, pH 7.8, 3 mM MgCl, 60 mM KCl, 6% glycerol, 60 mg/ml bovine serum albumin, and 1 µg/ml
dGdC. As indicated, both MgCl and KCl concentrations were
varied for certain experiments while leaving the other buffer
conditions unchanged. Mock incubations (as would be required for TBP
binding) were typically performed at 30 °C for 30 min. The
temperature of these incubations was also varied as indicated. Unless
otherwise noted, electrophoresis was in 0.7% agarose and 0.5 
TBE (1 TBE is 90 mM acid, 2.5 mM EDTA) for 3
h at 100 V.DNase I Footprinting
Reconstituted nucleosome core
particles were spun on a 5-20% sucrose gradient in TE plus 1
mM phenylmethylsulfonyl fluoride (20 h at 35,000 rpm in a
Beckman SW 41 rotor) to remove unreconstituted DNA. These were treated
as described above, scaling up 7-fold. Following incubation, samples
were digested with DNase I at a concentration of 12 µg/ml for 5 min
at room temperature. The reaction was stopped with a 2-fold excess of
EDTA, made 0.25% SDS and 0.3 M NaAc, and extracted with
phenol/chloroform and chloroform alone prior to precipitation.
Electrophoresis was on a 6% denaturing gel (Hayes et al.,
1990).
Nucleosome Core Dissociation Facilitates TBP
Binding
We first examined the stability of nucleosome cores that
contained a modified 5 S rRNA gene into which the adenovirus major late
TATA box (TATAAAAG) had been inserted (see ``Materials and
Methods''). The TATA box was positioned 81 bp from the dyad axis
of the nucleosome core (Pruss et al., 1995). Control
digestions with DNase I and micrococcal nuclease, which determine the
rotational and translational position of DNA relative to the histone
octamer, confirmed that these were unchanged from those obtained with
the wild type X. borealis 5 S rRNA gene (see Fig. 2). ()Our binding conditions were optimized to facilitate the
association of yeast TBP and TFIIA with the TATA box in naked DNA (Fig. 1, lane 2) (Horikoshi et al., 1989,
1992; Buratowski et al., 1989; Maldonado et al.,
1990). Ionic conditions were 20 mM HEPES/KOH, pH 7.8, 12
mM Tris-HCl, pH 7.8, 5.6 mM dithiothreitol, 60
µg/ml bovine serum albumin, 1 µg/ml dGdC, 3 mM MgCl, 60 mM KCl, and 6% glycerol. Addition of
reconstituted nucleosome cores (see ``Materials and
Methods'') to this solution at progressively lower concentration
leads to the progressive dissociation of the nucleosomes as revealed by
native polyacrylamide gel electrophoresis (Fig. 1, lanes
3-5). Note that because of dilution a smaller mass of DNA
and hence radioactivity is loaded in lane 5 compared with lane 3. At 3 ng/µl, histone octamer-associated DNA exceeds
free DNA by 3-fold (determined from the PhosphorImager), whereas
at 1 ng/µl, free DNA exceeds histone octamer-bound DNA by more than
2-fold (Table 1). Thus the relative proportions of nucleosomal
compared with free DNA change significantly simply through adjustment
of concentration. Control experiments indicated that DNA was not being
lost through nonspecific association with the reaction tube. These
involve measuring the low levels of radioactivity associated with the
reaction tube after the sample had been loaded on the gel or most
simply by removal of the solution from the tube following the dilution
process and measuring any residual radioactivity in the tube by
scintillation counting.
concentration is 7 mM. Lane 1 contains
G-specific cleavage reaction as a marker, lanes 2-4 contain
diluted core particles, and lane 5 is naked DNA. The
10-11-bp periodicity indicative of a rotationally phased
nucleosome is evident in lane 2 but becomes progressively more
like naked DNA in lanes 3 and 4.
10M. Nucleoprotein complexes and free DNA are indicated. Oct, octamer.
The Influence of the Histone Tails, Ionic Conditions, and
Temperature on Nucleosome Dissociation
Rigorous earlier work had
made use of analytical ultracentrifugation to determine the
thermodynamic parameters governing nucleosome dissociation using
nucleosome core particles containing a mixture of DNA sequences (Ausio et al., 1984a, 1984b). Ausio and colleagues also presented a
thermodynamic analysis of the existing data on this topic (Stacks and
Schumaker, 1979; Cotton and Hamkalo, 1981; Ausio et al.,
1984a, 1984b). Our results are in general agreement with this careful
analysis. Here we have also examined more qualitative aspects of
nucleosome dissociation using reconstituted nucleosome cores containing
the X. borealis 5 S rRNA gene. We have investigated the role
of the core histone tails and MgCl concentration,
parameters that influence transcription factor binding (Lee et
al., 1993; Vettesse-Dadey et al., 1994) but that had not
been the focus of previous studies. We present our results as gel
retardation assays that separate histone-bound from free DNA ( Fig. 3and Fig. 4).
concentration is 3
mM. B, effect of MgCl concentration on
tail-less octamer stability. Core particles with histone tails removed
incubated with different levels of MgCl. Lanes
1-5 contain no MgCl, and lanes 6-10 contain 5 mM MgCl. Histone-bound and free DNA
are indicated.
concentration,
temperature, and KCl concentration on the disruption of nucleosomes. A, MgCl concentration was varied from 0 mM (lanes 1-5) to 5 mM (lanes
6-10) to 12 mM (lanes 11-15) with
KCl concentration and temperature held constant as described under
``Materials and Methods.'' B, temperature of
incubation was changed from 0 °C (lanes 1-5) to 25
°C (lanes 6-10) to 37 °C (lanes
11-15) with MgCl and KCl concentrations held
constant. C, KCl concentration was varied between 70 and 280
mM as indicated. MgCl concentration was held at 5
mM, and the temperature was 30 °C. Histone-bound and free
DNA are indicated. Bar indicates the population of nucleosome
cores to which a second histone octamer is bound (Ausio et
al., 1984a, 1984b).
concentrations
are increased from 0 to 5 mM (Fig. 3B). In
contrast, nucleosome cores in which the histone tails are present are
progressively destabilized by an increase in MgCl
concentration from 0 to 5 and 12 mM (Fig. 4A, lanes 1-5, Table 2). Thus the histone tails and
MgCl concentration will significantly influence the
stability of histone-DNA interactions at dilutions of nucleosomes
commonly used in transcription factor binding experiments.
A promoter
(Ranjan et al., 1994) (
)(Fig. 5, lanes
1-5) and a dinucleosome containing two reiterated 5 S rRNA
genes (Ura et al., 1995) are also destabilized by dilution (Fig. 5, lanes 6-10). These results are
indicative of the generality of this nucleosome disruption phenomenon
(Stacks and Schumaker, 1979; Cotton and Hamkalo, 1981; Ausio et
al., 1984a, 1984b).Conclusions
We describe experiments that make use
of standard conditions for examining the binding of trans-acting
factors to DNA. Due to the sensitivity of the available gel shift and
DNase I footprinting assays, together with inherent limitations in the
availability of specific trans-acting factors and potential chromatin
remodeling complexes, these assays typically utilize DNA at very low
concentrations. We demonstrate here that the concomitant dilution of
nucleosomes leads to loss of histone-DNA contacts under conditions that
retain the binding of trans-acting factors.
)))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
ABSTRACT
This article has been cited by other articles:
|
|
 |
|
  A. Prasad, S. S. Wallace, and D. S. Pederson Initiation of Base Excision Repair of Oxidative Lesions in Nucleosomes by the Human, Bifunctional DNA Glycosylase NTH1 Mol. Cell. Biol., December 15, 2007; 27(24): 8442 - 8453. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. W. Adkins, S. K. Williams, J. Linger, and J. K. Tyler Chromatin Disassembly from the PHO5 Promoter Is Essential for the Recruitment of the General Transcription Machinery and Coactivators Mol. Cell. Biol., September 15, 2007; 27(18): 6372 - 6382. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Griesenbeck, H. Boeger, J. S. Strattan, and R. D. Kornberg Affinity Purification of Specific Chromatin Segments from Chromosomal Loci in Yeast Mol. Cell. Biol., December 15, 2003; 23(24): 9275 - 9282. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. J. Cost, A. Golding, M. S. Schlissel, and J. D. Boeke Target DNA chromatinization modulates nicking by L1 endonuclease Nucleic Acids Res., January 15, 2001; 29(2): 573 - 577. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Howe and J. Ausió Nucleosome Translational Position, Not Histone Acetylation, Determines TFIIIA Binding to Nucleosomal Xenopus laevis 5S rRNA Genes Mol. Cell. Biol., March 1, 1998; 18(3): 1156 - 1162. [Abstract] [Full Text]
|
|
|
|
 |
|
 E. Y. Shim, C. Woodcock, and K. S. Zaret Nucleosome positioning by the winged helix transcription factor HNF3 Genes & Dev., January 1, 1998; 12(1): 5 - 10. [Abstract] [Full Text]
|
|
|
|
 |
|
 A. N. Imbalzano, G. R. Schnitzler, and R. E. Kingston Nucleosome Disruption by Human SWI/SNF Is Maintained in the Absence of Continued ATP Hydrolysis J. Biol. Chem., August 23, 1996; 271(34): 20726 - 20733. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |