(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 (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.
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 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.
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 10
M. Nucleoprotein complexes and free DNA are indicated. Oct, octamer.
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 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.
Figure 4:
Effect of MgCl 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).
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 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.
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 TRA 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).