(Received for publication, March 26, 1997, and in revised form, May 5, 1997)
From the Departments of Molecular Biology and
Oncology and ¶ Internal Medicine, University of Texas Southwestern
Medical Center, Dallas, Texas 75235-9140
Nuclease hypersensitive sites exist in
vivo in the chromatin of the integrated human immunodeficiency
virus (HIV)-1 proviral genome, in the 5-long terminal repeat (LTR)
within the promoter/enhancer region near Sp1 and NF
B binding sites.
Previous studies from the Kadonaga and Jones laboratories have shown
that Sp1 and NF
B can establish hypersensitive sites in a truncated
form of this LTR when added before in vitro chromatin
assembly with Drosophila extracts, thus facilitating
subsequent transcriptional activation of a linked reporter gene upon
the association of additional factors (Pazin, M. J., Sheridan, P. L.,
Cannon, K., Cao, Z., Keck, J. G., Kadanaga, J. T., and Jones, K. A. (1996) Genes & Dev. 10, 37-49). Here we assess the role of
a full-length LTR and 1 kilobase pair of downstream flanking HIV
sequences in chromatin remodeling when these transcription factors are
added after chromatin assembly. Using Xenopus laevis oocyte
extracts to assemble chromatin in vitro, we have confirmed
that Sp1 and NF
B can indeed induce sites hypersensitive to DNase I,
micrococcal nuclease, or restriction enzymes on either side of factor
binding sites in chromatin but not naked DNA. We extend these earlier
studies by demonstrating that the process is ATP-dependent
when the factors are added after chromatin assembly and that histone
H1, AP1, TBP, or Tat had no effect on hypersensitive site formation.
Furthermore, we have found that nucleosomes upstream of NF
B sites
are rotationally positioned prior to factor binding and that their
translational frame is registered after binding NF
B. On the other
hand, binding of Sp1 positions adjacent downstream nucleosome(s). We
term this polar repositioning because each factor aligns nucleosomes
only on one side of its binding sites. Mutational analysis and
oligonucleotide competition each demonstrated that this remodeling
required Sp1 and NF
B binding sites.
In its most fundamental form, packaging of DNA in the nucleus of a eukaryotic cell into chromatin starts with the creation of the polynucleosomal chain. Each nucleosome consists of a core particle consisting of the histone octamer and 146 bp1 of DNA wrapped in the left-handed superhelical sense, and a linker region of variable length but generally less than 50 bp, which interacts with histone H1 and/or other accessory proteins (reviewed in Ref. 1). This polynucleosomal structure is further looped and folded into various higher order structures, leading to the functional sequestration of the genome in the interphase nucleus at a DNA concentration of approximately 50 mg per ml (reviewed in Refs. 2 and 3).
The packaging of DNA into chromatin adds levels of complexity to the problem of transcriptional regulation. Nucleosome formation often hinders the accessibility of transcription factors to occupy their binding sites within promoters (reviewed in Refs. 4-6). On the other hand, formation of the nucleosome can in some instances increase the affinity or action of factor binding (7-9). In addition, transcriptional elongation is attenuated by the formation of the polynucleosomal chain, leading to further potential regulatory opportunities (reviewed in Ref. 10). Although it seems clear that the eukaryotic transcription apparatus has evolved to work through chromatin and not simply naked DNA, we are only now beginning to develop an understanding of these mechanisms (reviewed in Ref. 11).
Windows in chromatin that have already bound transcription factors, or that will allow their binding, are operationally detected as nuclease hypersensitive sites (reviewed in Ref. 12). Several studies have focused on the mechanism of how these sites are generated. Theoretically one might imagine that DNA replication might be required for transcription factors to invade nucleosomes at the time of chromatin reassembly (13), but the only evidence for this mechanism that has thus far been obtained deals with the establishment of transcriptional repression (14, 15 and references therein). Rather, DNA replication-independent mechanisms seem to have evolved to allow transcription factors access to DNA sequences complexed to histone octamers. At least three different "protein machines" have been identified, by either biochemical or genetic experiments (or both), that utilize ATP to facilitate loading of transcription factors onto promoters originally packaged as nucleosomes: (i) the SWI·SNF complex (16; reviewed in Refs. 17 and 18); (ii) NURF (nucleosome remodeling factor) (19, 20); and (iii) RSC (remodeling the structure of chromatin), a complex at least 10-fold more abundant than SWI·SNF in yeast (21).
We report here studies on chromatin remodeling within the HIV-1 5-LTR
where the viral promoter and enhancer are located. We selected this
system because the LTR has been well characterized with respect to
transcription factor binding sites (reviewed in Refs. 22-24) and
nuclease hypersensitive sites in chromatin (25, 26). Furthermore,
recently the laboratories of Kadonaga and Jones have shown that Sp1 and
NF
B can establish hypersensitive sites in a 0.42-kbp truncated-LTR
linked to a reporter gene when added before in vitro
chromatin assembly with Drosophila extracts (27). Here we
have taken advantage of these findings to explore further the mechanism
for hypersensitive site formation and the potential for repositioning
of nucleosomes along a full-length LTR, including 1 kbp of downstream
HIV sequences, when factors are added either before or after chromatin
assembly. Using Xenopus laevis oocyte extracts to assemble
chromatin in vitro, we have confirmed that Sp1 and NF
B
can indeed establish nuclease hypersensitive sites, even when added
after chromatin assembly. We demonstrate that the process is
ATP-dependent, that nucleosomes upstream of NF
B sites
are rotationally positioned, and that factor binding leads to a novel
polar repositioning of flanking nucleosomes.
The HIV-1
SphI/SphI fragment of pHXB2 (28) was inserted
into pNEB193 (lacking a AvaI polylinker site). The newly
generated plasmid termed pWLTR11 contained nucleotides 1-1456 of the
HIV-1 genome (5-LTR and 5
fragment of the gag gene) and
was 4202 bp in length. Plasmids pWLTR12 and pWLTR13 containing
mutations within Sp1 and NF
B binding sites, respectively (29), were
constructed similarly. Fig. 1 shows the positions of
factor binding sites, key restriction enzyme sites, and the sequences
mutated. Single site labeling of circular DNA utilized the procedure of
Shimamura et al. (30). Briefly, plasmids were linearized at
a single cutting restriction enzyme site and treated with alkaline
phosphatase. DNA was then purified by phenol/chloroform extraction
followed by ethanol precipitation and labeled with
[
-32P]ATP using T4 polynucleotide kinase. DNA circles
were generated in a T4 DNA ligase-catalyzed reaction. To remove
radioactive label from non-ligated molecules, material was treated with
alkaline phosphatase and DNA was purified by phenol/chloroform
extraction followed by ethanol precipitation. Labeled DNA was dissolved
in extraction buffer (see below) at 10 ng/µl and stored at
80 °C (used within 1-2 weeks without signs of significant radiolysis or
nicking).
Preparation of Oocyte Extract and Assembly of Minichromosomes
Xenopus laevis oocyte extracts were
prepared according to Shimamura et al. (30). Briefly,
ovaries from adult frogs were treated for 3 h at room temperature
with 0.15% collagenase type II (Sigma) in buffer OR-2 (5 mM HEPES, 1 mM Na2HPO4,
82 mM NaCl, 2.5 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, pH 7.6).
Dispersed oocytes were then washed with 10 changes of OR-2 buffer.
Small oocytes were discarded, and large stage-6 oocytes were gently
washed with three changes of ice-cold extraction buffer (20 mM HEPES, 5 mM KCl, 1.5 mM
MgCl2, 1 mM EGTA, 10% glycerol, 10 mM -glycerophosphate, 0.5 mM dithiothreitol,
pH 7.6). An equal volume mixture of oocytes and extraction buffer was
centrifuged in a Beckman SW55Ti rotor for 90 min at 40,000 rpm at
4 °C. The interphase between the pellet and upper lipid layer was
aliquoted and stored at
80 °C. The protein concentration of
extracts typically was about 3 mg/ml. Assembly and analyses of
radioactive plasmid into chromatin was done according to the protocols
of Worcel and co-workers (30-33), in a final volume 10-50 µl. A
10-µl reaction mixture consisted of 4 µl of oocyte extract, 15-25
ng of labeled DNA (the optimal amount was experimentally determined for
each DNA preparation), 10 ng of creatine phosphokinase, and 1 µl of
10 × chromatin assembly buffer (400 mM creatine
phosphate, 20 mM ATP, 10 mM MgCl2).
All reagents were dissolved in extraction buffer (final pH 7.0). In some experiments chromatin was assembled in the presence of total histone H1 isolated from human placenta, final concentration 0.7 ng/µl (that was about 2 histone H1 molecules per nucleosomal repeat). The mixture was then incubated for 5 h at 27 °C. Where
indicated, chromatin was assembled in the presence of recombinant human
transcription factors. Sp1, p50, and c-Jun were purchased from Promega.
p65 and p50/p65 were produced in a baculovirus expression system using standard techniques (34). p65 was 6xHis-tagged, which allowed us to
purify the p65 homodimer and p50/p65 heterodimer using
Ni2+-NTA-agarose columns (Qiagen). TBP and Tat were
produced as glutathione S-transferase-fusions in a bacterial
expression system and purified as described previously (35). In some
experiments, factors were added to DNA 15 min before the oocyte extract
was added. Final concentrations of the factors in the assembly mixtures
were as follows: 10 nM Sp1, 50 nM NF
B (homo-
or heterodimer), 50 nM AP1 (c-Jun homodimer), 50 nM TBP, and 100 nM Tat. In other experiments Sp1 (20 nM) and NF
B (100 nM) were added
after chromatin assembly, and the reaction was allowed to proceed for
another 30 min. ATP was removed from reaction mixtures that contained
assembled minichromosomes according to Tsukiyama et al.
(36); mixtures were either treated with apyrase (final concentration
0.01 unit/µl) for 15 min at 27 °C or filtered through spin columns
containing Bio-Gel A-1.5m (Bio-Rad).
Supercoils introduced into DNA circles upon minichromosome formation were analyzed by agarose gel electrophoresis. Aliquots of assembly reactions were mixed with 0.5 volume of stop solution (0.6% SDS, 50 mM EDTA, and 6 mg/ml proteinase K) and incubated for 1 h at 50 °C. Gel loading dye buffer was added, and samples were then run on 1.25% SeaKem agarose gels (1 × TAE) for about 12 h at 5 V/cm at 4 °C. Alternatively, electrophoresis was performed in the presence of 15 µg/ml (30 mM) chloroquine. Some DNA samples were electrophoresed in two dimensions. After the first dimension, gels were soaked in chloroquine-containing buffer, turned 90°, and run in a second dimension. When electrophoresis was completed, gels were fixed in 1% cetyltrimethylammonium bromide, dried, and exposed to x-ray films or PhosphorImager screens. To analyze the nucleosomal ladders of minichromosomes, CaCl2 (final concentration 3 mM) and micrococcal nuclease (Worthington, final concentration 0.15 unit/µl) were added to the assembly mixture. Digestion proceeded 1-90 min at room temperature and was stopped by adding 0.5 volume of stop solution (see above). After incubation at 50 °C samples were run on 1.5% SeaKem agarose gels (1 × TAE), and gels were fixed, dried, and exposed as described above.
Nuclease Hypersensitivity Site AnalysesChromatin was assembled using plasmids labeled at the PmeI restriction site. After assembly was completed, CaCl2 and MgCl2 (final concentration 3 and 5 mM, respectively) were added and then chromatin was digested with DNase I (Worthington, final concentration 8 units/ml) or micrococcal nuclease (MNase) (Worthington, final concentration 4 units/ml) for 2 or 4 min at 27 °C. In parallel experiments, naked DNA was digested with DNase I (0.04 unit/ml) or micrococcal nuclease (2 units/ml). Digestion was terminated by adding stop solution. After incubation for 1 h at 50 °C, DNA was purified by phenol/chloroform extraction followed by ethanol precipitation. Solubilized DNA samples were then digested with EcoRI and electrophoresed on 0.5% SeaKem, 2% NuSieve agarose gels (1 × TAE). Before analyzing for the accessibility of restriction sites, NaCl and MgCl2 (final concentration 50 and 7 mM, respectively) were added to assembled minichromosomes. Restriction enzyme digestion (final concentration 1 unit/µl) was for 7, 15, or 30 min at 27 °C. Reactions were terminated by adding stop solution, and DNA samples were purified as above, digested with EcoRI, and electrophoresed on 1.5% SeaKem agarose gels (1 × TAE). Gels were fixed, dried, and exposed to x-ray films and PhosphorImager screens as described above. Radioactivity in restriction fragments was quantified using ImageQuant software (Molecular Dynamics).
DNase I Footprinting AssayChromatin was assembled using plasmids labeled at either the BglII or AvaI restriction sites. After assembly was completed, minichromosome and naked DNA samples were digested with DNase I as described above. Purified DNA was digested either with BglII and SstI or with AvaI and ScaI. Heat-denatured DNA was run on 8 M urea, 6% polyacrylamide sequencing gels. Gels were dried and exposed as described above.
Gel Mobility Shift AnalysisThe oligonucleotides used in
gel mobility shift assays were 66-mer duplexes spanning NFB and Sp1
binding sites (
111 to
46 bp), with either wild type or mutated
sites. Binding reactions between end-labeled double-strand probes (200 pmol) and purified proteins (500 pmol) were carried out for 30 min at
27 °C in a final volume of 20 µl of binding buffer (20 mM HEPES, 50 mM NaCl, 1 mM
dithiothreitol, 0.5 mM EDTA, 3 mM
MgCl2, 10% glycerol, 0.05% Nonidet P-40, pH 8.0) in the
presence of different amounts of specific unlabeled competitors.
Samples were run on native 5% polyacrylamide gels (0.25 × TBE)
at 10 V/cm at 4 °C. Gels were fixed, dried, and exposed as described
above.
To
characterize the kinetics and efficiency of chromatin assembly, we
assayed for the production of negative DNA supercoils with time of
incubation in Xenopus extracts by separating resulting purified DNA circles on agarose gels in the absence or presence of
chloroquine (Fig. 2, A and B). The reactions
reached completion after 5 h of assembly at 27 °C. To determine
the writhe for fully assembled molecules we performed two-dimensional
electrophoresis in the presence of a standard that contained an array
of topoisomers differing in linking numbers by single integers. As
shown in Fig. 2C, the number of negative DNA
supercoils introduced into the 4.2-kbp circles exhibited a distribution
with an average between 23 and
24. Assuming 1 negative supercoil
per nucleosome (2), these circles possessed nucleosomes about every 175 to 185 bp. To investigate further the assembly process we digested the
fully assembled minichromosomes with micrococcal nuclease (MNase) and estimated the DNA lengths of the purified DNA products by gel electrophoresis. As shown in Fig. 2D, the size of the
protected mononucleosome DNA fragments is close to 146 bp, as expected
for core particles, whereas oligonucleosomes exhibit repeat lengths between 165 and 175 bp, with an inter-band background indicative of
slightly irregular spacing. Taken together, the DNA supercoiling and
nucleosome ladder results indicate that the Xenopus oocyte system works to assemble bona fide chromatin in our hands.
ATP-dependent Induction of DNase I-hypersensitive Sites in the HIV-1 Promoter in Chromatin by Sp1 and NF
To determine
if Sp1 and NFB could create DNase I-hypersensitive sites within the
HIV 5
-LTR in the Xenopus chromatin assembly system like
they can in the Drosophila chromatin assembly system (27),
these recombinant proteins were added individually or together before
or after chromatin assembly. As shown in Fig. 3A, when added before chromatin assembly individually, each
protein created hypersensitive sites apparently flanking both sides of their DNA binding sites within the LTR, and when added together the
hypersensitive sites, which were centered at positions
40 and
160
bp (large arrowheads), exhibited a broadened intervening footprint (for purposes of presentation, unless otherwise stated, the
term NF
B will refer to the p50/p65 heterodimer). NF
B also created
hypersensitive sites in flanking vector sequences in a region
coincidentally exhibiting a 9/11 bp match to its consensus binding site
(small arrowheads). It is significant that all of these
sites are chromatin-specific and do not appear when these factors are
complexed with naked DNA (Fig. 3B), a point not addressed by
the earlier study (27). Furthermore, if chromatin was first assembled
in the absence of exogenous Sp1 and NF
B, the later addition of these
factors induced the hypersensitive sites in an
ATP-dependent fashion (Fig. 3C, asterisks) (for
purposes of presentation, when factors are added after chromatin
assembly they are designated with asterisks). Although three different forms of NF
B (p50/p65, p50/p50, and p65/p65) each stimulate in vitro transcription from the HIV enhancer on naked DNA (37, 38),
the p50/p50 form cannot activate transcription in vitro on
chromatin templates (27). However, we found that all three of these
forms of NF
B were active in forming DNase I-hypersensitive sites
when added either before or after chromatin assembly, in the presence
or absence of Sp1 (data not shown; see below). We also tested
recombinant forms of other transcription factors (TBP, AP1(c-Jun), and
Tat) and found that these proteins (either alone or in combination with
Sp1 and NF
B) did not create any chromatin structural alterations
(Fig. 3A and data not shown). We conclude that Sp1 and
NF
B are key players in chromatin remodeling within the HIV 5
-LTR in
the Xenopus system just as they are in the
Drosophila system (27).
DNase I-hypersensitive Site Formation by Sp1 and NF
To gain further evidence that Sp1
and NFB must bind to their target sites in the HIV 5
-LTR to create
hypersensitive sites, we repeated the experiments shown in Fig. 3 but
with DNA constructs that possessed base substitution mutations in the
corresponding binding sites known to inhibit transcription in
vivo (29) (see Fig. 1C). When DNA containing mutated
Sp1-binding sites was mixed with Sp1 in the absence of NF
B and
chromatin was assembled, hypersensitive sites characteristic for Sp1
binding were not induced (Fig. 4). However, as expected,
NF
B was still able to create its characteristic hypersensitive sites
in this mutant construct in the presence or absence of Sp1 (Fig. 4).
When DNA containing mutated NF
B-binding sites was mixed with Sp1 and
assembled into chromatin, the specific pattern of DNase I
hypersensitivity characteristic for Sp1 was present but weaker as
compared with that of the wild type control, suggesting that the
GC-rich nature of wild type NF
B sites may help preserve Sp1
occupancy after chromatin assembly (Fig. 4). NF
B was still able to
create weak hypersensitive sites spaced even broader than those
characteristic for NF
B binding per se, even though its
binding sites had been mutated, presumably because residual half-sites
were still present in this mutant and secondary binding occurred
through sequence similarities between NF
B and Sp1 binding sites
(Fig. 4; see Fig. 1C). Gel mobility experiments using a
66-mer duplex spanning these mutations verified that this sequence
still had residual affinity to bind NF
B (data not shown). Interestingly, when both factors were combined with the construct carrying the mutated NF
B sites and chromatin was assembled,
hypersensitive sites characteristic (although somewhat weaker) of the
wild type construct were generated (Fig. 4). Apparently the known
cooperative interactions between Sp1 and NF
B (39, 40) leads to
synergism in occupancy of the weakened binding sites. We also performed competition experiments with 33-mer duplex oligomers containing wild
type NF
B- or Sp1-binding sites (or both). The presence of both
competitors completely abolished the formation of these
hypersensitive sites, while either alone ablated the formation of its
target hypersensitive sites (data not shown).
Rotational Positioning of Nucleosomes before and after Occupancy of Sp1 and NF
To investigate the possibility of
rotational positioning of nucleosomal DNA on the surface of histone
octamers as well as prove that Sp1 and NFB actually target their
binding sites in the LTR when added either before or after chromatin
assembly, we separated DNase I digests on sequencing gels. When NF
B
bound to naked DNA it protected from
109 to
77 nt corresponding to its two binding sites (non-coding strand), whereas Sp1 protected from
87 to
64 nt corresponding to the highest affinity binding site of
the triplet array (41) (Fig. 5A). Binding of
both factors gave the expected additive footprint spanning from
109
to
64 nt, in either naked DNA or chromatin (Fig. 5, A and
B). Very similar footprints were seen on chromatin
substrates with the three different forms of NF
B whether the factors
were added before or after nucleosome assembly (Fig. 5B;
data not shown). We conclude that these transcription factors can
target their binding sites in chromatin. In contrast to the
similarities in the footprinted regions in these factor binding sites
between naked DNA and chromatin, the chromatin but not naked DNA
samples exhibited approximately a 10-nt periodicity in DNase I cutting
upstream of
220 nt (Fig. 5, B and C, closed circles), even in the absence of added factors, immediately
adjacent to a short footprinted region spanning
205 to
220 nt (Fig.
5, B and C, X), suggesting the
presence of rotationally positioned nucleosomes. It is striking that
this presumptive rotational positioning extends beyond the limits of
the 5
-LTR (
454 nt) onto flanking vector sequences (Fig.
5C, closed circles). In contrast to these results, no rotational positioning was observed downstream of Sp1
binding sites, in the presence or absence of Sp1 or NF
B (or both)
(Fig. 5B, data not shown).
Polar Repositioning of Nucleosomes by Sp1 and NF
To
determine if nucleosome positions within the LTR are modulated by Sp1
and NFB binding when the factors are added either before or after
chromatin assembly, we mapped the positions of MNase cutting sites. In
the absence of these factors, almost all MNase cutting sites were
common between the naked DNA controls and chromatin samples
(Fig. 6, lanes 11-13 and 18-20),
indicating that the nucleosomes were not translationaly positioned
along the DNA sequence. However, binding of Sp1 and NF
B to DNA prior to chromatin assembly generated a pair of major MNase-hypersensitive sites in positions essentially identical to the DNase I-hypersensitive sites that flank factor binding sites (Fig. 6A, lanes 7-9;
large arrowheads); these sites were not generated upon
factor binding to naked DNA controls (Fig. 6B, lanes
14-16). In addition, Sp1 and NF
B generate minor
MNase-hypersensitive sites at +140 and
340 bp, respectively (Fig.
6A, lanes 3-6; closed circles, small arrowheads). Interestingly, these minor sites correspond to
nucleosome length distances from the major hypersensitive sites,
suggesting that binding of Sp1 and NF
B each position adjacent
nucleosomes, one upstream of NF
B binding sites and the other
downstream of Sp1 binding sites. Consistent with this interpretation is
the observation that cutting sites in the regions of these putative positioned nucleosomes centered at positions
290 and +40 bp become protected when factors bind (Fig. 6A, lanes 3-6; open
circles, bars). It is significant that these same major and minor
hypersensitive sites and footprints are generated when factors bind
after chromatin assembly (Fig. 6C, compare lanes
21-23 with lanes 24-26; closed circles, arrowheads, open
circles, and bars). Furthermore, MNase ladder
experiments reveal that nucleosomes indeed reside on sequences downstream of the Sp1 binding sites (minichromosomes labeled at BglII site located at +20 bp, data not shown). We conclude,
therefore, that Sp1 and NF
B each remodel chromatin structure by
repositioning nucleosomes with polarity.
Quantitative Assessment of Hypersensitive Site Formation Using Restriction Enzymes
To more accurately judge the increase in
nuclease accessibility generated by Sp1 and NFB binding before or
after chromatin assembly, we digested minichromosomes to completion
with several different restriction enzymes and determined the cleavage
efficiencies. As shown in Fig. 7, binding of Sp1 to DNA
before chromatin assembly increased the accessibility of the
PvuII site located at
21 bp downstream of Sp1-binding
sites but did not affect the efficiency of cleavage at the
ScaI site located at
140 bp upstream of NF
B-binding sites. However, binding of NF
B to DNA before chromatin assembly increased the accessibility at both these sites. Maximal cutting at
both sites occurred when both Sp1 and NF
B were added prior to
chromatin assembly (Fig. 7) or when Sp1 was added prior to and NF
B
was added after chromatin assembly (data not shown). Adding both
factors after chromatin assembly (Fig. 7) or NF
B prior to followed
by Sp1 after chromatin assembly (data not shown) were less efficient in
increasing accessibility. Cleavage results obtained at this
ScaI site were very similar to those exhibited at the
AvaI site at
158 bp (data not shown). Furthermore, no significant differences were observed when the different forms of
NF
B were used in similar experiments (data not shown). In addition,
increased restriction site accessibility was lost as expected when
constructs containing mutated binding sites were used or when ATP was
depleted prior to adding factors to chromatin assembled on the wild
type construct (data not shown). Similar studies at numerous other
sites revealed no increase in accessibility upon factor binding (see
Fig. 7 legend), except for an increase in cutting at HinfI
(+124 bp) caused by Sp1 (data not shown), correlating well with the 3
border of the positioned nucleosome detected by MNase cleavage
experiments (Fig. 6). In conclusion, the results obtained with
restriction enzymes are in complete agreement with the results obtained
using both DNase I and MNase as probes for chromatin structure.
Our results on how the Xenopus system designs or
remodels the chromatin structure of the HIV-1 promoter/enhancer and
flanking regions in response to human transcription factors are
schematically presented in Fig. 8. In the absence of
transcription factors, the nucleosomes along the HIV-1 5-LTR are
rotationally positioned upstream of
220 bp (R ovals) but
randomly positioned elsewhere. Binding of NF
B results in
translational positioning of these upstream nucleosomes while still
maintaining rotational positioning (RT ovals), while binding
of Sp1 translationally positions a downstream nucleosome (T
oval). Each factor creates hypersensitive nuclease cutting sites
on either side of their binding sites in chromatin but not in naked
DNA. Together these factors create a local chromatin structure that
resembles the in vivo state (see below).
Our study extends the work of the laboratories of Kadonaga and Jones
who used Drosophila extracts for chromatin assembly
experiments (27). We have confirmed that Sp1 and NFB create
hypersensitive sites and have been able to examine repositioning of
nucleosomes on flanking bona fide HIV sequences that was not studied
previously. We show that this process is ATP- and target
site-dependent, and we have quantitated site generation
using restriction enzymes. In the Kadonaga, Jones, and co-workers'
study (27), chromatin structural analysis was only performed when
factors were bound to DNA before chromatin assembly, not after; in
addition, nucleosomes were already positioned downstream of Sp1 binding
sites on the flanking vector sequences in the absence of this
transcription factor, and no change in positioning was detected upon
prebinding Sp1 per se; this is not the case in the
Xenopus system with HIV sequences, because Sp1 binding, even
after chromatin assembly, leads to polar repositioning of downstream
nucleosome(s). The sensitivity of our studies also permitted observing
rotational positioning upstream of NF
B sites not looked for in the
earlier work. Finally, repositioning of nucleosomes by NF
B per
se was not studied previously, which we found here also to exhibit
polarity.
Statistical positioning theory predicts that occupied factor binding
sites would create boundary constraints that would exclude nucleosomes
and passively lead to positioning on both sides of the boundary (42,
43). However, we observed positioning only downstream of occupied Sp1
sites and only upstream of occupied NFB sites. Furthermore, in other
in vitro chromatin assembly experiments using the mouse
immunoglobulin gene, we have observed the same polarity of nucleosome
positioning with respect to the orientation of the NF
B binding site
adjacent to the intronic enhancer (data not shown). This polarity
suggests that specific segments of these transcription factors interact
with specific segments of the histones. This positioning is absolutely
precise with respect to the DNA helix for the rotationally positioned nucleosomes upstream of NF
B sites (Fig. 8, RT ovals).
Possibly the footprinted region at
205 to
220 nt (Fig. 5; Fig. 8,
black lines below) sets up this positioning because of a
need for a specific exposed helix surface to bind factors in the oocyte
extracts; this footprinted sequence is AT-rich and has been reported to bind NF-AT (23, 24) as well as nuclear matrix protein(s) (44). The
interacting Xenopus protein(s) remains to be identified as well as the significance, if any, of such rotational positioning.
Chromatin structure appears to be a major control point in regulating
HIV-1 gene expression. In vivo analysis has revealed hypersensitive sites near Sp1 and NFB binding sites for integrated provirus exhibiting low basal expression, similar to but apparently somewhat broader than the zone described in our studies, in cell lines
in which NF
B is thought not to have been activated (25, 26). Perhaps
the transient activation of this factor can create sites that become
"memorized" after factor disappearance, and this chromatin
structure is templated to daughter cells; alternatively, the promoter
region possesses a battery of other factor binding sites, including
TFIID, LEF-1, Ets-1, and USF (see Fig. 1A). Binding of some
of these factors may be responsible for generating and/or broadening
the hypersensitive site in vivo (45), whereas AP-3-like, DBF1, possibly together with Sp1 binding may create another downstream site not seen in our studies (46).
Previous in vitro transcription studies on chromatin
templates have shown that the p65 form of NFB, which possesses a
transactivation domain, is required for recruitment of the basal
machinery for synergistic transcriptional activation with Sp1 (27). In
addition, direct protein interactions between Sp1 and p65 have been
identified and shown to be required for efficient activation of HIV
transcription (39, 40). Interestingly, we found that Sp1 binding to DNA before chromatin assembly potentiated the ability of NF
B to generate hypersensitive sites after chromatin assembly and observed that Sp1
enhanced the ability of NF
B to bind to partially mutated NF
B
sites (Fig. 4).
Activation of HIV-1 infected T-cells leading to subsequent increases in
HIV-1 transcription results in rearrangement or displacement of the
repressing nucleosome immediately downstream of the transcription start
site (26). Our in vitro assembly conditions did not result in this disruption. Further analysis will be needed to determine whether this change in chromatin structure is involved in activation of
transcription by the HIV-1 transactivator Tat. Nucleotide level resolution revealed that the region surrounding the TATA box did not
become footprinted after chromatin assembly in the presence of a
mixture of transcription factors (data not shown), indicating that
TFIID complexes did not form over the core promoter. It is possible
that Dr1, a TBP-binding repressor present in Xenopus oocytes, may account for this fact (47). In addition, we have found
that including -amanitin (final concentration 0.1 µM)
had no effect on hypersensitive site formation when Sp1 and NF
B were added either before or after minichromosome formation (data not shown),
thus arguing that hypersensitive site formation occurs independent of
transcription.
An interesting unanswered question is whether histones still remain
bound within the hypersensitive region of the HIV-1 5-LTR after
ATP-dependent nucleosome disruption by factor binding. In a
model in vitro system Owen-Hughes et al. (16)
have shown that the SWI·SNF complex can displace histones from a
GAL4-occupied hypersensitive site and that the hypersensitivity
remained even after removal of bound transcription factors by
oligonucleotide competition. We attempted related experiments in the
Xenopus system with the HIV LTR, where we performed gel
mobility analysis on the hypersensitive region after its excision by
restriction enzymes from purified minichromosomes. Although the
hypersensitive region exhibited a gel mobility indistinguishable from
naked DNA associated with Sp1 and NF
B, after oligonucleotide
competition this complex was converted into others and not simply to
either naked DNA or nucleosome bands (data not shown).
The Xenopus oocyte extracts used in our chromatin assembly
experiments contain only an embryonic form of histone H1, termed B4
(48), whose binding to nucleosomes is insufficient to repress the
transcription of certain genes that can be repressed by adult histone
H1 homologs during development (49). Therefore, we have repeated many
of these experiments with chromatin assembled with added histone H1 and
found similar remodeling whether the factors were added before or after
assembly, by DNase I-hypersensitive site analysis, MNase footprinting
assays, and by the observation of disruption of the MNase ladder for
minichromosomes labeled at the AvaI site located at 158
bp, and by noting an increase in accessibility to restriction enzyme
sites at
21 bp (PvuII) and
140 bp (ScaI)
(data not shown). This is in confirmation with the results of Kadonaga,
Jones, and co-workers (27) who used histone H1 containing chromatin.
Thus, even though histone H1 is known to be functionally important in
repressing gene expression and reducing nucleosome mobility (49, 50),
the ATP-dependent remodeling activity in oocyte extracts
was fully capable of overriding any potential inhibition by this
protein. Clearly, the nature of the remodeling activity remains to be
elucidated but may be amenable to study in these extracts.
We thank Dr. David Gross for a critical
review of this work; Drs. Yu Liu and Rolf Joho for Xenopus
oocytes; Drs. Missag Parseghian and Barbara Hamkalo for human histone
H1; Drs. Frank Mercurio and Migeal Barbosa for baculovirus expression
vectors encoding human NFB subtypes; and Dr. Kathy Potter for
baculovirus expression.