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(Received for publication, September 12, 1995; and in revised form, October 24, 1995) From the
A DNA-bending sequence has been used for in vitro reconstitution of nucleosomes in order to direct a nuclear factor
1 (NF-1) binding site into different nucleosome positions. By this
strategy nucleosomes were obtained that had one of two rotational
positions of the NF-1 binding site, one oriented toward the periphery
and the other toward the histone octamer, translationally positioned 50
and 45 base pairs, respectively, from the nucleosome dyad. The affinity
of partially purified NF-1 for these nucleosomal targets was compared
with its affinity for free DNA by dimethylsulfate methylation
protection and DNase I footprinting assays. The binding affinity of
NF-1 to all nucleosomal targets was reduced 100-300-fold compared
with its affinity for free DNA. The two rotational settings of the NF-1
site showed the same binding affinity for NF-1 as did other nucleosome
constructs in which the NF-1 binding site was translationally
positioned from 10 to 40 base pairs from the nucleosome dyad. We
conclude that the nucleosomal inhibition of NF-1 binding is an inherent
characteristic of NF-1 since another transcription factor, the
glucocorticoid receptor, is able to bind to its DNA site in a
nucleosome.
The nuclear factor 1 (NF-1) ( The NF-1 protein family binds DNA
as dimers, and their binding sites are homologous to the partially
palindromic sequence
TTGGC(N) NF-1 binding sites are often
positioned close to the binding site of other transcription factors in
the regulatory region of many genes. In the retroviral MMTV promoter,
an NF-1 binding site is localized in the -76/-60 DNA
segment(1, 3) , next to the -185/-79 DNA
segment that contains four binding sites for the glucocorticoid
receptor (GR)(22) . Transcription from the MMTV promoter is
controlled by glucocorticoid hormone(23) , but the
glucocorticoid-induced transcriptional response is reduced about 5-fold
by a mutation in the NF-1 binding site(3) . This
hormone-dependent NF-1 enhancement might be explained by the
observation that the NF-1 protein is not bound to its binding site in
the silent MMTV promoter and that NF-1 binding depends on hormone
activation of the promoter(24) . This suggests that the
constitutive DNA-binding protein NF-1, which is located in the cell
nucleus, is prevented from binding to DNA by the specific chromatin
organization in the MMTV promoter. In line with this hypothesis,
chromatin remodelling of the MMTV promoter was shown to occur within
minutes of hormone stimulation (25) and to involve the
rearrangement of a positioned nucleosome covering the
-250/-60 DNA segment(26) , where GR and NF-1 bind.
Further studies of the chromatin structure of the MMTV promoter in
different cell lines and at different hormonal states have confirmed a
strong correlation between NF-1 promoter occupancy and the degree of
chromatin opening, the latter as measured by in situ restriction enzyme access(27, 28) . In
vitro studies involving the reconstitution of the GR and NF-1
binding DNA segment of the MMTV promoter into a nucleosome demonstrated
that the DNA was rotationally positioned into a preferred rotational
frame on the histone octamer surface. These studies showed that
purified glucocorticoid receptor protein could bind to its target
sequences in the nucleosome (29, 30, 31) while NF-1 was unable to bind to
its nucleosomal target(30, 31) . In these studies the
NF-1 binding DNA segment was rotationally positioned such that its two
consecutive major grooves containing the recognition sequence for NF-1
faced the histone octamer(30) . Thus, there remain two
explanations for the absence of NF-1 binding in the nucleosomally
reconstituted MMTV promoter(32) : (i) the NF-1 protein is
incapable of binding to its site in nucleosomal DNA irrespective of the
nucleosomal positioning, or (ii) the inability of the NF-1 protein to
bind is due to the particular position of the NF-1 site on the histone
octamer in the MMTV promoter (see above). These two explanations have
implications for how chromatin organization may restrict access of
certain transcription factors. This restriction in access may in turn
influence how gene induction is triggered by an inducible transcription
factor such as GR and a constitutive DNA binding transcription factor
such as NF-1. Here we address this issue by use of in vitro reconstituted nucleosomes in which a single NF-1 binding site is
held in various well defined rotational and translational positions
relative to the histone octamer. This positioning is achieved by
placing the NF-1 binding site within a segment of DNA-bending
sequence(33) . Our results show that the NF-1 protein binds
with at least a 100-fold lower affinity to a nucleosomal DNA site than
a corresponding site on free DNA. The rotational and translational
positioning of the binding site on the nucleosome had no effect on the
affinity of NF-1 for DNA. This is in contrast to GR, which binds to a
nucleosomal glucocorticoid response element (GRE) with a high affinity
if held in certain translational (34) and rotational (35) positions. Thus, the lack of NF-1 binding to a nucleosome
is an intrinsic property of the NF-1
Figure 1:
DNA sequences used for construction of
nucleosome probes. A, the DNA building blocks used for
construction of the 161-bp nucleosome probes. Only the top strand is
shown of the double-stranded DNA, in all cases flanked by asymmetric AvaI sites used for unidirectional ligation. Letters in boldface represent the NF-1-binding
-77/-63 DNA segment of the MMTV promoter. The NF-1
half-sites in No4 and Ni4 are indicated by arrows. The diamond represents the dyad of the NF-1 site. Stars indicate G residues protected from dimethylsulfate methylation by
NF-1 binding. B, the 161-bp DNA segments No4 and Ni4 are
shown. These and all other constructs consisted of 95 bp of TG motif (TG, filled bar), 15 bp of NF-1 binding site (open box) positioned either in a facing out (No) or a facing
in (Ni) configuration, and 51 bp of flanking vector sequence (thin
line). Also shown is a summary of DNase I footprinting data from Fig. 2A, showing the nucleosomal 10-bp DNase I cutting
pattern with black triangles for the upper strand and open
triangles for the lower strand. Arrows signify the first
nucleosome-induced exonuclease III stop on each strand as obtained from Fig. 3A (black arrow for the top strand and open arrow for the bottom strand).
Figure 2:
Rotational positioning of nucleosomal
No4 and Ni4. A, free (free) and nucleosomal (Nuc) DNA were digested with DNase I. Top strand of No4 (lanes 1-3), bottom strand of No4 (lanes
4-6), top strand of Ni4 (lanes 7-9), and
bottom strand of Ni4 (lanes 10-12) are shown. Vertical arrows indicate the partially palindromic NF-1
half-sites. Triangles indicate nucleosomally induced DNase I
cuts (black triangles for the top strand and open
triangles for the bottom strand). G+A, G + A
sequencing lane. B, graphic representation of the distribution
of DNase I-cut sites in nucleosomal No4 and Ni4 for both bottom and top
strand. This illustrates the rotational setting of the No4 and Ni4
site, highlighted in black in the DNA helix, in relation to
the DNA major groove and the histone protein
surface.
Figure 3:
Translational positioning of nucleosomal
No4 and Ni4. A, exonuclease III protection analysis. Top
strand (lanes 1-6 and 13-18) and bottom
strand (lanes 7-12 and 19-24) of free
No4, nucleosomal No4, free Ni4, and nucleosomal Ni4 were digested with
1.5 units of enzyme for increasing times. The first nucleosomally
induced stop in each strand is indicated by an arrow. The NF-1
half-sites are indicated by vertical black arrows. G+A = G + A sequencing lane. B,
translational positioning of the nucleosomal No4 and Ni4 according to
exonuclease III analysis. The distance from the dyad of the NF-1 site
to the nucleosome dyad is indicated.
Rat liver nuclear extracts were used
as an alternative source of NF-1 protein. Rat liver nuclei were
purified as described before(39) . The nuclear pellet was then
extracted in 10 ml of buffer C (20 mM HEPES, pH 7.6, 25% (v/v)
glycerol, 0.42 M NaCl, 1.5 mM MgCl
The rotational positioning of the NF-1
site in each reconstituted nucleosome was determined by DNase I
footprinting. DNase I is known to cleave the DNA in the minor groove
with higher efficiency where DNA is bent away from the enzyme cleavage
site. Rotationally positioned nucleosomal DNA will thus generate a
10-bp DNase I ladder(40, 41) . When we compared
nucleosomal No4 and Ni4 DNA with their free counterpart there was a
10-bp periodicity of DNase I cutting with intervening segments of
protection in the nucleosomal DNA (Fig. 2A). The staggering
of DNase I cutting between the two strands was 2-4 bp, as
expected for DNA that is wrapped around a histone octamer(42) .
Both No4 and Ni4 gave the same histone-induced 10-bp periodicity,
indicating that they adopt the predicted rotational position: the major
grooves of the NF-1 half-sites in No4 face the periphery, while the
major grooves of NF-1 half sites in Ni4 face the histone octamer
surface (Fig. 2B). The only differences between the DNase I
cutting pattern of nucleosomal No4 and that of nucleosomal Ni4 were
seen in the region containing the NF-1 binding site. These differences
are due to different DNA sequences being located in the minor groove
facing the periphery. The translational positions of the NF-1 sites
in the two mononucleosomes were determined by exonuclease III
protection analysis. Nucleosomal No4 and Ni4 gave similar exonuclease
III patterns, showing that they adopt the same translational
positioning (Fig. 3A). Full-length probe (position 161) was
partially protected from exonuclease III digestion of the top strand in
reconstituted nucleosomes for both No4 and Ni4. The first protected
base on the bottom strand of nucleosomal No4 was at position 18, giving
a 144-bp segment of protected DNA, as expected for nucleosomal DNA
located in one strongly preferred translational position. The
additional exonuclease III protections are caused by the previously
described capacity of exonuclease III to digest DNA within a
nucleosome, which gives rise to the characteristic 10-bp ladder (43) seen in Fig. 3A. In nucleosomal Ni4 the first
stop in the bottom strand occurred at position 19. We conclude that
nucleosomal No4 and Ni4 adopt the same rotational and translational
position of the DNA-bending segment. The dyads of the NF-1 sites are
positioned 50 and 45 bp from the nucleosome dyads in No4 and Ni4,
respectively (Fig. 3B), in opposite rotational settings (Fig. 2B).
Figure 4:
NF-1/DNA binding monitored by
dimethylsulfate methylation protection analysis. A, free No4 (lanes 1-4), nucleosomal No4 (lanes 5-8),
free Ni4 (lanes 9-12), and nucleosomal Ni4 (lanes
13-16) were exposed to dimethylsulfate in the absence or
presence of NF-1 protein (NF-1). The dimethylsulfate-induced
methylation was revealed by cleaving with piperidine. The amount of
NF-1 is given in units (U), where 1 unit is the concentration
of NF-1 required for 50% protection of an NF-1 site on free DNA in
DNase I footprinting. Stars indicate G residues protected from
methylation by NF-1. Vertical arrows indicate NF-1 half-sites. Thick arrows indicate the reference bands used to normalize
the variation in loading and the extent of dimethylsulfate methylation
in each lane for quantitative analysis. B, data from
experiments as in panel A displayed as NF-1-dependent
dimethylsulfate methylation protection (in percentage of control) as a
function of the logarithmic concentration of NF-1 protein in units.
Each point in the diagram is the mean of three experiments. Standard
deviations are indicated for each point. n, nucleosomal; f, free.
DNase I
footprinting was also used to assay binding of NF-1 to free and
nucleosomal DNA. A distinct footprint of NF-1 bound to free DNA was
seen both for No4 and Ni4 (Fig. 5A, lanes 2-5 and 11-14). NF-1 bound to the corresponding nucleosomal DNA
also gave protections over the NF-1 binding site both in No4 (Fig. 5A, lanes 7-9) and Ni4 (Fig. 5A, lanes 16-18). Quantification
by PhosphorImager analysis of the NF-1 dependent DNase I protection
showed that an approximately 300-fold higher concentration of NF-1 was
required to obtain 50% protection of a nucleosomal NF-1 site than was
required to obtain 50% protection of a binding site located on free
DNA. In agreement with the dimethylsulfate methylation protection data,
there was no difference in NF-1 binding affinity between nucleosomal
No4 and Ni4 (Fig. 5B, compare nNo4 with nNi4). DNase I
footprinting analyses of nucleosomal DNA also showed that NF-1 induced
protection and hypersensitivity outside of the NF-1 binding region.
This is probably caused by nonspecific interactions between protein and
DNA since it also occurred in control experiments using nucleosomal DNA
lacking an NF-1 site.
Figure 5:
NF-1-DNA binding monitored by DNase I
footprinting analysis. A, bottom strand of free No4 (lanes
2-5), nucleosomal No4 (lanes 6-9), free Ni4 (lanes 11-14), and nucleosomal Ni4 (lanes
15-18) were digested with DNase I in the absence or presence
of NF-1. Amount of NF-1 is given in units (U; for unit
definition see legend to Fig. 4A). Lanes 1 and 10 are sequencing lanes (G+A) for No4 and Ni4,
respectively. Arrows indicate NF-1 half-sites, and open
boxes indicate the DNase I footprint induced by NF-1. Filled
circles show NF-1-induced DNase I-hypersensitive sites outside the
NF-1 binding site, and empty circles show NF-1-induced DNase I
protections outside the NF-1 binding site. Thick arrows indicate the reference bands used to normalize the variation in
loading and the extent of DNase I digestion in each lane for
quantitative analysis. B, data from experiments as in panel A displayed as NF-1-dependent protection of DNA cutting
by DNase I (in percentage of control) as a function of the logarithmic
concentration of NF-1 protein in units. Each point in the diagram is
the mean of three experiments. Symbols are as in Fig. 4B.
The results described above were obtained
using recombinant NF-1 protein expressed in vaccinia virus. A series of
similar binding experiments were also performed using NF-1 protein that
was partially purified from rat liver nuclear extracts. These extracts
showed the same relative difference in affinity between a nucleosomal
NF-1 site and a free NF-1 site (data not shown).
Figure 6:
Nucleosome probes for studies of NF-1
binding to a nucleosomal site with different translational positioning.
The 161-bp DNA fragments, with the thick line indicating the
95-bp-long TG motif and the open box indicating the 15-bp NF-1
site. The ellipsoid indicates the translational positioning of
the histone octamer according to exonuclease III analysis. The dashed ellipsoid indicates an alternative nucleosome
positioning. The distance from nucleosome dyad to dyad of NF-1 binding
site is given at the right. All four constructs have an NF-1
site that is oriented with its major grooves toward the periphery
according to DNase I footprinting analysis.
We have shown for the first time that NF-1 cannot bind to its
binding site within a nucleosome irrespective of the rotational and
translational position of the binding site. The inability of NF-1 to
bind to nucleosomal DNA is characteristic of NF-1. GR is perfectly
capable of binding to a single GRE in the same nucleosome context as we
have used in this study(34, 35) . The GR binding
affinity to a nucleosomal GRE is often 2-3-fold lower than its
free counterpart. There are, however, certain nucleosomal GRE positions
where GR cannot bind. These positions are well defined, and the effects
depend on the topology. For example, a GRE at the nucleosome dyad has
high affinity for GR when oriented toward the periphery but becomes
inaccessible when moved 5 bp relative to the TG motif(35) . In
the latter position, the major groove is rotated into the opposite
direction, i.e. oriented toward the histone octamer. The
inhibition of NF-1 binding to a nucleosomal binding site is in sharp
contrast to GR binding to a nucleosomal GRE. Our binding data do not
allow us to exclude the possibility that the nucleosome structure is
locally perturbed during the NF-1 binding reaction. Thus it remains to
be shown whether NF-1 is at all able to form a specific ternary complex
with an intact nucleosome. Experiments that examine the protection from
dimethylsulfate methylation have the advantage of detecting close
protein-DNA contacts, and are thus highly specific. However, these
experiments do not allow insight into the nucleosomal structure of the
bound complex since dimethylsulfate methylation is uneffected by
histone-DNA interaction. Conversely, the DNase I footprinting
experiments reveal the typical 10-bp DNase I ladder, which is induced
by the nucleosome structure, which should allow the parallel evaluation
of specific NF-1 binding and nucleosome structure. However, the high
concentration of NF-1 protein, which is required for DNase I protection
in a nucleosomal NF-1 site also affects DNase I digestion outside of
the NF-1 binding site. This is probably due to nonspecific binding of
NF-1 to DNA and possibly the binding of other contaminating proteins
present in our partially purified NF-1 preparations. This results in a
progressively increased DNase I protection at the NF-1 site and a
concomitant fading of the nucleosome-induced DNase I pattern. Thus it
is not possible to determine whether the nucleosomes that generate the
10-bp ladder also contain one specifically bound NF-1 protein dimer.
Nevertheless, we can still conclude that NF-1 has an at least 100-fold
lower affinity for a nucleosomal NF-1 site than it has for an NF-1 site
in free DNA. If the DNA organization in the nucleosome must be
perturbed for NF-1 binding to occur, then the stability of each
nucleosome, as determined by its DNA sequence, would be expected to
affect NF-1 binding affinity. Although translational nucleosome
positioning has been described in several promoters (for a review see (41) ), little is known about the precision of this positioning
at the base pair level(44) . Our finding that the NF-1 protein
cannot bind to a nucleosomal target 10-50 bp from the dyad shows
that a precise nucleosome arrangement is not needed for the binding
inhibition to occur. However, our experiments do not determine where
the inhibition occurs relative to the nucleosome border. Attempts to
position the NF-1 site further than 50 bp away from the nucleosome dyad
failed due to problems with variability in translational position of
the histone octamer caused by use of longer TG motif DNA. Cordingley et al.(24) have observed that NF-1 occupancy of the
MMTV promoter in vivo depends on the glucocorticoid hormone.
This dependence can be simply explained by our results, which show that
nucleosomal DNA is selectively inaccessible for NF-1 but not for
GR(34, 35) . The modes of DNA binding of NF-1 and GR
relative to histone/DNA arrangements in a nucleosome may explain the
difference in selectivity. GR is known to form specific DNA contacts
with two consecutive major grooves on the same side along the DNA
axis(45, 46, 47) , apparently without any
effect on the DNA structure(47) . The NF-1 protein also forms
specific DNA contacts with two consecutive major grooves along one side
of the DNA length axis(10) . However, in contrast with GR, NF-1
binding to DNA has been reported to enhance a preformed bend in the DNA
segment that flanks its binding site in the adenovirus terminal
repeat(48) . Whether it is the NF-1-induced DNA structure,
steric hindrance of the NF-1 protein as such, or some other feature of
the NF-1 Since NF-1 binding
sites occur in many viral and cellular regulatory DNA segments, it is
likely that the inhibition of NF-1 binding that is induced by the
nucleosome is of functional significance also in other inducible
promoters. One such case might be the CYP1A1 gene, where an Ah
receptor, which binds dioxin, induces chromatin opening and NF-1
binding(9) . As described previously for the MMTV
promoter(26) , the uninduced CYP1A1 promoter also
contains positioned nucleosomes, some of which are perturbed by the
dioxin-induced promoter activation(51) . Another example is the
liver-specific serum albumin enhancer, which contains three
specifically positioned nucleosomes in liver cells where it is
active(8) . One of the three nucleosomes is structurally
perturbed and hypersensitive to DNase I. This perturbed nucleosome
contains an NF-1 site and binding sites for liver-specific factors such
as HNF3. The albumin enhancer is only occupied by NF-1 in liver cells
where the enhancer is active, even though NF-1 is present in many
tissues. McPherson et. al.(8) suggest that
liver-specific factor(s) might induce the chromatin structure required
for NF-1 to gain access to its binding site. A role of chromatin in
gene regulation has been demonstrated in yeast cells by genetic
interruption of histone H4 synthesis. This resulted in derepression of
several previously silent genes(52) . Likewise, titration of
the histone pool in Xenopus oocytes by coinjection of
competitive DNA resulted in a 10-fold decrease in nucleosome density of
injected DNA. A parallel transcriptional activation of the MMTV
promoter occurred independent of the glucocorticoid
receptor(53) . These results suggest that one function of
chromatin is to keep constitutively DNA binding transcription factors,
such as NF-1 and TATA box binding protein, away from their targets in
promoters that should be kept silent. Such a chromatin-dependent effect
might be important for two reasons: (i) to avoid transcriptional
leakage in promoters that should be inactive, and (ii) to reduce
protein-DNA interaction in general and thereby direct the constitutive
DNA binding transcription factors to the active promoters. This
repressive function of chromatin suggests that induction of a
previously silent promoter would require another class of transcription
factors. This other class of factors would, in contrast to NF-1,
possess the capacity to bind nucleosomal targets and to mediate
chromatin remodelling. The remodelling would allow entry of the
constitutively DNA binding factors. In this model, a gene that is
reversibly regulated between a silent and an active state requires that
the nucleosome binding activity of the inducing transcription factor be
controllable, for example by ligand binding. This control mechanism has
been seen for the GR and the Ah receptor.
Volume 271,
Number 1,
Issue of January 5, 1996 pp. 153-159
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)family of DNA binding
proteins can act both as transcription factors for RNA polymerase II
genes (1) and as initiation factors for viral DNA replication
in adenovirus-infected cells(2) . NF-1 family members act as
transcriptional activators in regulatory regions of several genes of
both viral and cellular origin, such as the mouse mammary tumor virus
(MMTV) promoter(1, 3) , the human papilloma virus type
16 enhancer(4) , the adipocyte-specific P2
enhancer(5) , the Xenopus laevis vitellogenin B1
gene(6) , the proenkephalin gene of human brain(7) ,
the liver-specific serum albumin enhancer(8) , and the CYP1A1 gene(9) .
GCCAA(10, 11) . Several different
forms of NF-1 are found in various differentiated cell
types(12) . They originate from the expression of four
different NF-1 genes(13) , different splicing variants (13, 14) and covalent modifications such as
glycosylation (15) and phosphorylation(16) . The NF-1
dimers are stable and seem to be formed cotranslationally, and
different NF-1 protein variants are able to
heterodimerize(17) . However, it is not known whether
heterodimers occur in vivo. The various NF-1 proteins often
mediate transcriptional activation but may also participate in the
repression of genes in certain cellular contexts (18, 19) . The N-terminal domain, sufficient for DNA
binding and dimerization, of all NF-1 proteins is highly conserved and
contains four cysteine residues that are strictly required for DNA
binding(20) . This DNA-binding domain is not homologous to any
of the other known classes of DNA-binding motifs such as the zinc
fingers, leucine zippers, or helix-turn-helix
motifs(11, 21) .
DNA complex. These findings
suggest how the organization of DNA into nucleosomes can be exploited
in the cell to create DNA binding hierarchies for various classes of
transcription factors.
DNA Constructs
The construction of various
plasmids containing a single transcription factor binding site within
repeats of DNA-bending sequences has been described
previously(34) . Briefly, the plasmid pNo4 was constructed by
consecutive cloning of a 30-bp DNA segment containing an NF-1 binding
site, followed by four 20-bp DNA segments consisting of a DNA-bending
sequence (referred to as the TG motif(33) ) into the asymmetric AvaI site of pGem-Q2(34) . Another construct, pNi4,
differed from pNo4 in that the NF-1 binding site was moved 5 bp
relative to the periodicity of the DNA-bending sequence (Fig. 1). No and Ni oligonucleotides contain the NF-1 site from
the mouse mammary tumor virus promoter, at positions -77 to
-63 (3) , and they contain 15 bp of DNA-bending
sequence(33) . We generated plasmids containing a 161-bp EcoRI/HindIII DNA insert, which was used for
nucleosome reconstitution. This insert contained 157 bp of
double-stranded DNA and four nucleotides of 5`-protruding
single-stranded DNA at each end. The first nucleotide in the top
strand, the EcoRI site, was given number 1. (
)
Nucleosome Reconstitution
DNA
labeling and fragment isolation were carried out as
previously(34) . Both nucleosome reconstitution with salt
dilution and the following purification by glycerol gradient
centrifugation were performed as described previously (29) with one modification, namely the final NaCl concentration
after salt dilution was 0.13 M.Nuclease Protection and NF-1 Binding
Exonuclease
III, DNase I footprinting, and dimethylsulfate methylation protection
were performed as described (Refs. 34, 29, and 35, respectively).
Quantification of NF-1 binding was done with PhosphorImager analysis and ImageQuant
software version 3.3
(Molecular Dynamics).
Preparation of NF-1 Protein
Recombinant NF-1
protein was isolated from approximately 10
HeLa cells grown
in roller flasks and infected with vaccinia virus that contained a
full-length clone for NF-1 (36) with six histidines fused to
the N terminus (constructed and kindly provided by Drs. Jacky Schmitt
and Hendrik Stunnenberg at EMBL, Heidelberg). The cells were harvested
in phosphate-buffered saline using a rubber policeman, and subsequent
steps were carried out at 4 °C. Cells were washed in
phosphate-buffered saline and homogenized in 2 volumes of buffer A (10
mM Tris-HCl, pH 7.9, 5 mM MgCl, 10 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 0.2% aprotinin
(Trasylol), 0.7 µg/ml leupeptin, 0.7 µg/ml pepstatin, 0.5
mM 
-mercaptoethanol, 0.15 mM spermine, 0.5
mM spermidine) using a glass homogenizer (Kontes Glass Co.,
Vineland, NJ). The homogenate was transferred to a 50-ml centrifuge
tube, and 5 M NaCl was added to achieve a final concentration
of 0.4 M NaCl. The specimen was incubated for 20 min on ice
and then centrifuged at 27,000 g for 30 min at 4 °C using an SS-34
rotor in a Sorvall RC-5B centrifuge. The clear supernatant was
incubated in a 50-ml Falcone tube with 2.5 ml of
Ni-NTA-agarose (Qiagen), equilibrated with buffer B
(20 mM Tris-HCl, pH 8.0, 2 mM MgCl
, 1
mM phenylmethylsulfonyl fluoride, 5 mM -mercaptoethanol, and 10% glycerol (v/v)) containing 4 mM imidazole, for 150 min at 4 °C under constant mixing. The
matrix was then transferred and packed into a small plastic column and
washed with two column volumes of buffer B containing 4 mM imidazole and subsequently washed with two column volumes of
buffer B containing 15 mM imidazole and with two column
volumes of buffer B containing 30 mM imidazole. The
recombinant NF-1 protein was then eluted with a linear gradient of
30-300 mM imidazole in buffer B with a total gradient
volume of 60 ml. The amount of active NF-1 protein was determined in
each fraction by DNase I footprinting. The peak fractions were pooled,
glycerol and dithiothreitol were added to 15% (v/v) and 1 mM,
respectively, and the specimen was stored at -110 °C. The
protein concentration in this preparation was less than 0.1
µg/µl(37) . We can estimate the purity of NF-1 protein
that actively binds to DNA in these preparations using the following
information: (i) 50% specific DNA binding in a DNase I footprinting
assay (see below) is obtained with 10 nl of the preparation, and (ii)
NF-1 binds DNA as a homodimer and has a molecular mass of 56.8 kDa (36) and a dissociation constant of about 2 
10M(38) for specific DNA
binding. We conclude that the purity of these preparations is at least
10% (w/w). One unit of NF-1 binding activity is defined as the amount
of NF-1 protein required to saturate a specific DNA site to 50% in a
DNase I footprinting assay containing 1.5 fmol of free DNA in an
incubation volume of 40 µl.
, 0.2
mM NaEDTA, 1 mM phenylmethylsulfonyl
fluoride, and 0.5 mM dithiothreitol) for 30 min at 0 °C
with occasional stirring, followed by centrifugation at 25,000 g in an SS-34 Sorvall rotor at 4 °C for 30 min. The clear
supernatant was applied onto a 50-ml Sephadex G25 column (Pharmacia
Biotech Inc.) equilibrated in buffer D (20 mM Tris-HCl, pH
7.6, 10% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride)
containing 0.1 M KCl, and the macromolecular fraction was
collected according to volume calibration with 10 ml of blue
dextrane (Pharmacia). The pool from the G25 column
was clarified by centrifugation at 17,000
g in an
SS-34 Sorvall rotor at 4 °C and then applied onto a 3.5-ml
double-stranded DNA-cellulose column, equilibrated with buffer D
containing 0.1 M KCl, at 0.12 ml/min, and then washed with two
column volumes of buffer D containing 0.1 M KCl and then with
two volumes of buffer D containing 0.15 M KCl. NF-1 was eluted
with buffer D containing 0.35 M KCl. The salt concentration of
the eluate was adjusted to 0.1 M KCl by the addition of buffer
D without salt and then applied onto a 1-ml Mono Q column
(Resource, Pharmacia) equilibrated with buffer D
containing 0.1 M KCl. The column was eluted with a linear
gradient of KCl from 0.1 M to 0.55 M in buffer D,
total gradient volume 21 ml. NF-1 protein, assayed by DNase I
footprinting, was eluted around 0.26 M KCl. Dithiothreitol was
added to 5 mM, and NF-1 was stored at -110 °C.
Rotational and Translational Positioning of an NF-1
Binding Site in Mononucleosomes Reconstituted in Vitro
Based on
previous experience (34) we constructed DNA fragments that were
161 bp long and contained a single NF-1 binding site. The NF-1 site was
placed within a synthetic DNA-bending sequence having a 10-bp
periodicity of (A/T)
NN(G/C)NN, referred to as
the TG motif (33) (Fig. 1). This DNA sequence is known
to direct rotational setting of DNA on a histone octamer such that A/T
segments are located at sites of minor groove compression and G/C
segments at sites of major groove compression(33) . We used
this bending sequence to direct the rotational positioning of the 15-bp
NF-1 binding DNA segment into two opposite rotational frames: No4, with
the two consecutive major grooves of the two NF-1 half-sites facing
toward the periphery of the nucleosome, and Ni4, with the two major
grooves of the two half-sites facing toward the histone octamer protein
surface (compare No and Ni in Fig. 1A). These DNA
fragments contain the NF-1 binding sites (15 bp), 95 bp of TG-bending
sequence, and 51 bp of flanking vector sequence (Fig. 1B).Low Affinity of NF-1 for Its Nucleosomal Binding
Site
Binding of NF-1 to DNA was measured by its ability to
protect the N-7 position of two guanosines from methylation by
dimethylsulfate. The binding affinity of NF-1 for an NF-1 site on free
DNA was compared with its nucleosomal counterpart in both rotational
orientations, No4 and Ni4. NF-1 protects a double G located 4 bp from
the dyad on both strands of the NF-1 site from methylation by
dimethylsulfate (10) (Fig. 1A). Considerably
higher concentrations of NF-1 protein were required to give partial
protection from methylation for nucleosomal No4 and Ni4 than were
required for free No4 and Ni4 (Fig. 4A). Only the bottom
strand analysis is shown, but the result was the same for the top
strand. Quantification of the dimethylsulfate methylation protection by
PhosphorImager analysis (Fig. 4B) showed that a 100-fold
higher NF-1 concentration was required to obtain 50% protection of the
nucleosomal NF-1 site than was required for same site located in free
DNA. NF-1 protects nucleosomal No4 and Ni4 to the same extent (Fig. 4B, compare nNo4 to nNi4).
Effect of Translational Position on NF-1 Binding to
Nucleosomal DNA
The dyad of the NF-1 site in No4 is located 50
bp from the dyad of the nucleosome. We investigated whether moving the
NF-1 site relative to the nucleosome dyad would have any influence on
NF-1 affinity for nucleosomal DNA. An additional four DNA segments were
constructed and reconstituted in vitro into nucleosomes. The
position of the NF-1 site and the affinity of NF-1 for the site were
measured as described above. These constructs and their nucleosomal
organization are shown in Fig. 6: 1No3, where the dyad of the
NF-1 site was located 30 bp from the nucleosome dyad; 2No2, where the
dyad of the NF-1 site was located 10 bp from the nucleosome dyad; 3No1,
where the dyad of the NF-1 site was located 20 bp from the nucleosome
dyad; and 4No, where the NF-1 site was located either 40 or 30 bp from
the nucleosome dyad (two alternative translational positions with the
same rotational position). In all of these constructs the same extent
of nucleosome-induced inhibition of NF-1 binding was observed as was
observed for No4 and Ni4 (data not shown).
DNA complex that inhibits NF-1-nucleosome interaction
remains to be investigated. We note that the TATA box binding protein
is unable to bind DNA in a nucleosome(49) . In that case the
reason may be the requirement of TATA box binding protein to form a
sharp kink in DNA upon binding(50) .
))
We thank Ulla Björk for skillful
technical assistance. We are grateful to Drs. Jacky Schmitt and Hendrik
Stunnenberg (EMBL, Heidelberg) for kindly providing recombinant
vaccinia strains for expression of NF-1 protein. We are indebted to
Kristina Nordström and Dr.
Björn Vennström for the help
and facilities for growing and harvesting HeLa cells infected with
vaccinia virus.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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