(Received for publication, August 14, 1995)
From the
Defined oligonucleosome model systems have been used to
investigate the molecular mechanisms through which the core histone
tail domains modulate chromatin structure. In low salt conditions, the
tail domains function at the nucleosome level to facilitate proper
organization of nucleosomal DNA, i.e. wrapping of DNA around
the histone octamer. Mg ions can substitute for the
tail domains to yield a trypsinized oligonucleosome structure that is
indistinguishable from that of an intact nucleosomal array in low salt.
However, Mg
-dependent formation of highly folded
oligonucleosome structures absolutely requires the histone tail
domains, and is associated with rearrangement of the tails to a
non-nucleosomal location. We conclude that the tail domains mediate
oligonucleosome folding and nucleosomal DNA organization through
fundamentally different molecular mechanisms.
Nucleosomal arrays, which consist of core histone octamer-DNA
complexes spaced at 200-bp
intervals, are the
fundamental nucleoprotein assembly of chromatin fibers and higher order
chromosomal domains. Nucleosomal arrays in various configurations also
are the substrates for both transcription and replication(1) .
Consequently, it has become important to elucidate the
structure-function relationships that pertain to nucleosomal arrays. At
the supranucleosomal level, it has long been known that nucleosomal
arrays exhibit a moderate degree of salt-dependent compaction, even in
the absence of linker
histones(2, 3, 4, 5, 6, 7, 8, 9) .
Recent studies have shown that the intrinsic folding of nucleosomal
arrays is more effective at repressing transcription initiation and
elongation by RNA polymerase III than the nucleosome per
se(10, 11) . Thus, oligonucleosome folding has
potential functional importance, both for the regulation of eukaryotic
gene expression, and presumably for other nuclear processes that
involve nucleosomal arrays as well.
The solution-state folding of
nucleosomal arrays is complex, and until recently has been poorly
understood. In 10-200 mM NaCl, nucleosomal arrays appear
to equilibrate between the extended beads-on-a-string conformation
exclusively present in low salt, and a partially folded structure that
is equivalent to a contacting zig-zag in its extent of
compaction(12, 13) . Furthermore, in 1-2 mM MgCl, regularly spaced nucleosomal arrays equilibrate
between the zig-zag-like conformation and a more highly folded
conformation that is equivalent to a 30-nm fiber in its extent of
compaction(14, 15) . While these observations
demonstrate that the core histones in and of themselves can direct
formation of highly folded chromatin structures, the mechanism(s)
through which the core histones function have yet to be identified.
Trypsinized oligonucleosomes lacking their core histone tail domains
remain unfolded in the presence of NaCl(8, 13) ,
suggesting that these domains in some way participate in core
histone-directed oligonucleosome folding. To identify the molecular
mechanism(s) of tail domain-mediated functions in chromatin, we have
used a combination of quantitative agarose gel electrophoresis (16, 17) and analytical ultracentrifugation to
determine the hydrodynamic shape, conformational deformability, and
surface charge density of trypsinized and intact nucleosomal arrays in
the presence and absence of MgCl. Results indicate a direct
role for the tail domains in in mediating both oligonucleosome folding
and proper wrapping of nucleosomal DNA; however, these functions are
mutually exclusive and occur through fundamentally different molecular
mechanisms. These observations imply that the tail domains rearrange in
chromatin in conjunction with separate actions at the nucleosomal and
supranucleosomal levels.
The R of nucleosomal arrays and DNA in each
gel was determined from their experimentally determined µ and
µ`
and the known P
using (16, 17) .
The data in Table 1also indicate that structures of native and trypsinized
nucleosomal arrays in low salt buffer are significantly different, as
was first reported by Garcia-Ramirez et al.(13) . In
particular, the decrease in s from
32 to 25 and the increase in R
from 26 to 32 nm in
E buffer confirm that the conformation of trypsinized nucleosomal
arrays in low salt is significantly more elongated than that of intact
arrays under the same conditions.
Figure 1:
Effect of MgCl on the
conformational deformability of trypsinized nucleosomal arrays.
Trypsinized 208-12 nucleosomal arrays (n = 12) in
either E (
) or E + 2.0 mM free MgCl
(
) were electrophoresed in 0.9-3.0% agarose gels and
analyzed as described under ``Experimental Procedures.''
Shown for comparative purposes are the results obtained previously for
208-12 DNA in E (dotted line)(16) , and intact n = 12 208-12 nucleosomal arrays in E (dashed line) (16) and E + 2.0 mM MgCl
(dotted
dashed line) (17) .
In 2.0 mM MgCl, however, the R
of
trypsinized nucleosomal arrays was independent of P
over the range of 40-150 nm, and indistinguishable from
that of intact nucleosomal arrays in low salt buffer. Together with the
data in Table 1, these results indicate that trypsinized
nucleosomal arrays in 2.0 mM MgCl
have the same
hydrodynamic shape and conformational deformability as intact
nucleosomal arrays in low salt.
In low salt E buffer, the µ of
saturated (n = 12) trypsinized nucleosomal arrays was
only 3% lower than that of the naked 208-12 DNA (Fig. 2). In 2.0
mM MgCl
, however, the µ
of
saturated trypsinized nucleosomal arrays was 20% lower than that of the
DNA (compare the n = 0 and n = 12
values in Fig. 3), indicating that Mg
ions are
binding to trypsinized nucleosomal arrays in conjunction with the
Mg
-dependent conformational change described above (Table 1; Fig. 1). Somewhat unexpectedly, the slopes of
the µ
versus n plots in 2 mM MgCl
were identical for trypsinized and intact
nucleosomal arrays containing
6 nucleosomes/DNA molecule. However,
although the slope of the trypsinized oligonucleosome plot in 2
mM MgCl
remained constant above n = 6 (Fig. 3), the slope of the intact nucleosomal
array plot changed markedly in this region. We have shown previously
that the additional decreases in µ
observed for n > 6 intact nucleosomal arrays in 2 mM MgCl
reflect the increased extents of oligonucleosome folding that
occur in proportion to the increased extent of nucleosome occupancy of adjacent 5 S repeats (17) .
Figure 2:
Effect of trypsinization on the
oligonucleosome µ in E buffer. Trypsinized 208-12
nucleosomal arrays in E (
) were electrophoresed in 0.2-0.7%
agarose gels and analyzed as described under ``Experimental
Procedures.'' Each data point represents the mean ±
standard deviation of four determinations. Also shown (
) are the
values obtained previously for intact 208-12 nucleosomal arrays in E (17) .
Figure 3:
Effect of trypsinization on the
oligonucleosome µ in 2 mM MgCl
.
Trypsinized 208-12 nucleosomal arrays in E + 2.0 mM MgCl
(
) were electrophoresed in 0.2-0.7%
agarose gels and analyzed as described under Experimental Procedures.
Each data point represents the mean ± standard deviation of four
determinations. Also shown (
) are the data obtained previously
for intact 208-12 nucleosomal arrays in E + 2.0 mM MgCl
(17) . The dashed line indicates
the linear regression through the trypsinized nucleosomal array data (r
= 0.98).
Our interpretation of the complex solution-state behavior of
trypsinized and intact nucleosomal arrays is schematically illustrated
in Fig. 4. Intact 208-12 nucleosomal arrays by numerous criteria
are best modeled as a fully extended structure in which two complete
turns of DNA are wrapped around each histone
octamer(12, 13, 25) . Sedimentation analyses
have demonstrated unequivocally that structural heterogeneity related
to partially unwrapped nucleosomal DNA (26) is not present in
the solution state in low salt conditions (12, 13, 14) . The µ of an
intact nucleosomal array in low salt is 20% lower than that of the
naked DNA, which is equivalent to 80-100 surface exposed positive
charges added by each histone octamer(16) . Finally, a
saturated 208-12 nucleosomal array in low salt is characteristically
less deformable than either naked 208-12 DNA or a subsaturated 208-12
nucleosomal array containing
1-2 nucleosome-free 5 S
repeats(16) .
Figure 4:
Schematic illustration of the influence of
MgCl on the solution state behavior of intact and
trypsinized 208-12 nucleosomal arrays. T represents the tail
domains; Mg represents Mg
ions.
In low salt buffer, trypsinized nucleosomal
arrays have markedly different structural properties than intact
nucleosomal arrays. A comparison of the µ of
trypsinized and intact nucleosomal arrays in E buffer (Fig. 2)
indicates that
85% of the surface positive charges of each histone
octamer are contributed by the lysine and arginine residues in the tail
domains. This is consistent with previous estimates derived from both
chemical modification (27) and thermodynamic (9) studies. Removal of the tails also leads to a decreased s
, an increased R
, and increased conformational deformability in
low salt. Each of these observations suggests that more linker DNA is
present in a trypsinized nucleosomal array (and therefore less DNA is
bound to the trypsinized octamer) in low salt. Taken together, our data
strongly support the previous conclusion by Ausio and colleagues (13) that only the central
100 bp of DNA is bound to each
trypsinized octamer within an array in the absence of salt.
Importantly, these data indicate clearly that under low salt conditions
the tails within a nucleosomal array function at the nucleosome level
to keep DNA at the nucleosome periphery wrapped around the histone
octamer.
In 2 mM MgCl, a trypsinized
nucleosomal array has both the same hydrodynamic shape and
characteristic conformational deformability as an intact nucleosomal
array in low salt (Table 1, Fig. 1). This demonstrates
that inorganic cations can mechanistically substitute for the tail
domains to organize the DNA at the periphery of the nucleosome. This
conclusion is supported by the previous findings that micrococcal
nuclease digests of trypsinized nucleosomal arrays are
indistinguishable from those of intact arrays in high salt (1
mM CaCl
) but produce mainly
100-bp products
in low salt (.05 mM CaCl
) (13) , and that
trypsinization does not influence the linking number of closed circular
nucleosomal arrays in 170 mM NaCl (28) . Our
observation that the µ
of saturated trypsinized
nucleosomal arrays in 2.0 mM MgCl
is 20% lower
than that of the naked 208-12 DNA molecule under the same conditions (Fig. 3) indicates that
40-50 Mg
ions, an amount equivalent to the total positive charges in the
tails, are taken up from the bulk solution concomitant with the
Mg
-dependent wrapping of DNA around the trypsinized
histone octamer. Importantly, despite having a structure that is
indistinguishable from that of an intact array in low salt, a
trypsinized nucleosomal array in 2.0 mM MgCl
is
incapable of folding (Table 1, Fig. 1; (14) ). We
therefore conclude that the core histone tails mediate oligonucleosome
folding through a mechanism that is distinctly different than the
coulombic-based DNA charge neutralization mechanism (29) involved in tail-mediated wrapping of nucleosomal DNA.
Do the tails remain bound to nucleosomal DNA in a folded nucleosomal
array? The answer to this question lies in the µ values
determined in 2.0 mM MgCl
. Studies to date have
identified four potential contributions to the µ
of an
intact 208-12 nucleosomal array in 2 mM MgCl
: the
surface negative charges of the naked DNA molecule, the positive
charges on the surface of the histone octamer(16) , nonspecific
binding of Mg
to naked DNA in 2.0 mM
MgCl
(17) , and Mg
that is
specifically taken up to organize nucleosomal DNA in the absence of
bound tails (Fig. 3). Nonspecific Mg
binding
to 208-12 DNA lowers the µ
by 25%, while the surface
positive charges in the histone octamer and Mg
uptake
during nucleosomal DNA wrapping each lower the µ
by
20%. Thus, any mechanism that leads to release of the tails from
nucleosomal DNA in 2 mM MgCl
(and concomitant
uptake of Mg
ions to replace the released tails) will
result in a µ
value that is the sum of all four
potential contributions, and hence 65% lower than the µ
of DNA in E buffer. By contrast, the µ
of both
trypsinized and intact nucleosomal arrays will be the same at any given n if the tails remain bound to nucleosomal DNA in 2
mM MgCl
(since no additional Mg
ions will be taken up by the intact arrays without tail release).
The data in Fig. 3indicate the µ
is identical
for both intact and trypsinized nucleosomal arrays in 2 mM
MgCl
, provided that n is
6. Thus, the tails
remain bound to nucleosomal DNA in 2 mM MgCl
if
the array is highly subsaturated and consequently
unfolded(17) . However, the µ
of a folded n = 12 intact nucleosomal array in 2 mM MgCl
(-0.80
10
cm
/V
s) is
67% lower than the µ
of naked DNA in E buffer
(-2.42
10
cm
/V
s),
indicating that the tails are released from their nucleosomal location
in 2.0 mM MgCl
concomitant with formation of
folded oligonucleosome structures. Both folding and release of the tail
domains require nucleosome occupancy of adjacent 5 S
repeats(17) . It is important to note that the tails do not
appear to be freely dissociated under salt conditions that
promote oligonucleosome folding(9) . Rather, the tails
presumably mediate oligonucleosome folding by interacting with
oligonucleosomal constituent(s) other than nucleosomal DNA. Potential
candidates include linker DNA (1, 30, 31) and/or neighboring core histone
components. An important implication of these results is that the tail
domains rearrange in chromatin in conjunction with their roles in
mediating structural transitions at both the nucleosomal and
supranucleosomal levels.