(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) ()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) .
The NF-1 protein family binds DNA
as dimers, and their binding sites are homologous to the partially
palindromic sequence
TTGGC(N)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) .
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-1DNA 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.
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, 0.2
mM Na
EDTA, 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.
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-1DNA 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) .
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.