Interaction of NF-E2 in the Human
-Globin Locus Control Region
before Chromatin Remodeling*
Yoshiaki
Onishi
and
Ryoiti
Kiyama
From the Research Center for Glycoscience, National Institute of
Advanced Industrial Science and Technology, AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
Received for publication, September 19, 2002, and in revised form, November 14, 2002
 |
ABSTRACT |
When transcription is initiated under repressive
conditions, such as when chromatin are packed together, binding
followed by the functioning of key components in the transcriptional
apparatus should be appropriately facilitated in the chromatin
architecture. We provide evidence that the erythroid-specific enhancer-
binding protein NF-E2 interacts with the cognate motif at DNase
I-hypersensitive site 2 of the human
-globin locus control region in
a repressive state. The nucleosome containing the NF-E2-binding
site showed characteristic rotational and translational phases in
vitro. The binding site had less affinity to the histone octamers
than nearby regions while showing greater accessibility to DNase I and
micrococcal nuclease. Furthermore, the motif was recognized by the
exogenous NF-E2 protein expressed in HeLa cells, which have a
repressive state of chromatin at the
-globin locus, as shown by
ligation-mediated PCR and chromatin immunoprecipitation assay. These
lines of evidence indicate that NF-E2 interacts with the cognate motif
on the nucleosome before chromatin is remodeled.
 |
INTRODUCTION |
The human
-globin locus
(
-LCR)1 spans 70 kb and
contains five developmentally regulated
-like globin genes, 5'-
,
G
, A
,
, and
-3', in that order. The expression of these
genes is restricted to erythroid tissues and regulated in a
developmental stage-specific manner (1). The human
-LCR confers high
level and tissue-specific expression to the
-globin genes and is
marked by erythroid-specific, DNase I-hypersensitive sites (HS-1 to
HS-4), which lie 6-18 kb upstream of the
-globin gene and function
together (2). In thalassemia DNA with large deletions encompassing the
LCR and the upstream sequence, expression of the downstream globin
genes is abolished, and the chromatin structure of the locus becomes resistant to DNase I cleavage (3), which suggests that the LCR mediates
activation of transcription of the genes at different stages in
development in conjunction with modification of the chromatin
structure. Transgenic mouse studies have also indicated that the LCR
provides an open, accessible chromatin structure regardless of where on
the genome the transgenic gene is integrated and can confer
position-independent expression to the integrated globin gene (4). In
addition to its enhancer activity, the LCR is important for
establishing the timing of DNA replication of the
-globin locus
during the S phase of the cell cycle (3, 5, 6). A central question in
developmental biology is how enhancers activate gene transcription in a
tissue- and developmental stage-specific fashion, a complex process
that takes place in the chromatin environment of the nucleus.
Most efforts to elucidate the function of the LCR have focused on
identifying the transcription factors, and many of the
erythroid-specific transcription factors with conserved binding sites
in the LCR have been clarified. These include NF-E2 (7, 8), GATA-1 (9-11), and erythroid Krüppel-like factor (12). Among them, NF-E2 plays a key role in the function of the LCR. NF-E2, a bZIP transcription factor, is a heterodimer of 45- and 18-kDa subunits. Expression of NF-E2 p45 is primarily restricted to erythroid cells, whereas NF-E2 p18 appears to be ubiquitously expressed. There appears
to be considerable functional redundancy among polypeptides that
recognize NF-E2 sites, and whereas the p18-p45 NF-E2 dimer itself may
be required to activate globin gene expression, other species may be
able to participate in the formation of the HSs (13). The overall
stimulatory activity of the LCR (at least of HS2) in the chromatin
environment of transgenic mice or in stably integrated constructs
appears to depend on NF-E2 motifs (14-18). From a series of studies
using transgenic mice with the
-globin yeast artificial chromosome,
it was suggested that the deletion of HS elements markedly reduced the
expression of all of the globin genes at all developmental stages
accompanying the malformation of DNase I hypersensitivity in the LCR
(19). In addition, it was also shown that the protein Bach1, which
heterodimerizes with the p18 NF-E2 subunit and interacts with the
Maf-responsive element (MARE) or NF-E2-binding site sequences at HS2,
HS3, and HS4, is able to cross-link HS sites, thereby looping out
intervening DNA regions (20). These results suggest that a series of
protein-protein and protein-DNA interactions establishes the formation
of a larger LCR complex (21). Furthermore, NF-E2 is critically involved in remodeling the nucleosome structure over the HS2 region, where it
interacts with the tandem MARE sites (22, 23). In contrast with the
study supporting a role for NF-E2 in
-globin gene expression, p45
NF-E2 null mice had nearly normal levels of
-globin protein (24),
and deletion of HS2 had no significant effect on the timing or extent
of expression of the gene (25). Recently, it was reported (26, 27) that
the chromatin-opening function of the LCR may not be the primary
activity at the endogenous mouse or human globin locus, because the LCR
can be deleted without affecting general sensitivity to DNase I. Although the mechanism of long range transactivation by the LCR is
still poorly understood, it is true that the LCR is required for
conferring high level globin gene expression throughout erythroid development.
Previously, we determined the nucleosome phases around HS2, and we
found that there are two major nucleosome positions in the HS2 region
in vivo (28). The
60 to +85 phase had a nucleosome dyad
axis at its center, where the tandem MARE sites were located, and
included the binding sites of the erythroid-specific transcription factors HS2NF5 and GATA-1. This phase was shown to be the only phase in
HeLa cells, and thus, this is the original state where NF-E2 first
interacts with the cognate motif. In contrast, the
100 to +45 phase
and several other minor phases were present in erythroid K562 cells. In
this state, the region containing HS2NF5 and GATA-1-binding sites was
partially or completely open resulting in an increased accessibility to
these factors. Furthermore, we also reported that the enhancer activity
at HS2 of the human
-LCR can be modulated by the curved DNA located
at a distance of two nucleosomes from HS2 and regulates nearby
nucleosome phases as a key nucleosome (29). In this paper, we
concentrated on characterizing the nucleosome structure over HS2, in
particular NF-E2 binding, and we demonstrated that NF-E2 interacts with
the cognate motif on the nucleosome before chromatin is remodeled.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
Restriction and modifying enzymes were purchased
from New England Biolabs (Beverly, MA). Cell culture materials were
obtained from Invitrogen. Other chemicals used were of the highest
quality commercially available and were purchased from Sigma.
Reconstitution of the Nucleosomes in Vitro--
Nucleosome core
particles were prepared from chicken erythrocytes as described
previously (28). After the preparation of core particles by gel
filtration through Sepharose CL-6B (Amersham Biosciences), the histone
octamers were prepared according to the method of Luger et
al. (30). Briefly, the core particles were applied to a
hydroxyapatite column equilibrated with 0.6 M NaCl, 5 mM Tris-HCl (pH 7.5), and 0.2 mM
2-mercaptoethanol, and the column was washed first with 0.65 M NaCl, 50 mM Na/PO4 (pH 6.0), 0.25 mM PMSF, and 0.5 mM dithiothreitol and then
with 65 mM NaCl, 50 mM Na/PO4, 0.25 mM PMSF, and 0.5 mM dithiothreitol. The octamer
was eluted with 2.5 M NaCl, 50 mM
Na/PO4 (pH 6.0), 0.25 mM PMSF, and 0.5 mM dithiothreitol and dialyzed against 10 mM
Tris-HCl (pH 7.5), 2 M NaCl, and 5 mM
2-mercaptoethanol. Nucleosomes were reconstituted onto DNA fragments
either by exchanging them with the DNA from the histone core particles
or by dialysis with the histone octamer.
Micrococcal Nuclease (MNase) Digestion Assay--
The
nucleosomes were reconstituted in vitro using the
end-labeled DNA fragments corresponding to the
100 to +45 and
60 to +85 nucleosome phases, respectively. MNase digestion was performed at a
final concentration of 0.025 units/ml at 25 °C for 2 and 5 min. DNA
was purified and electrophoresed on 4% polyacrylamide gels in 40 mM Tris acetate, 1 mM EDTA, and 5% glycerol.
Southern Blotting--
The nucleosomes were reconstituted
in vitro using a DNA fragment containing the entire HS2
region (
10,989 to
10,769 relative to the cap site of the
-globin
gene) and treated with MNase as described previously (28). The
~146-bp fragments were recovered from 6% polyacrylamide gels and
digested with restriction enzymes. DNA was electrophoresed on an 8%
polyacrylamide gel and transferred onto the membrane. Hybridization and
detection were performed as described previously (29). The probe was
the same DNA fragment used in nucleosome reconstitution.
Computer Analysis of the Three-dimensional Structure--
The
three-dimensional structure of the DNA curvature was predicted with
TRIF 1.00 software (sgjsl.weizmann.ac.il/usr/users/Curvature) as
described previously (31).
Electrophoretic Mobility Shift Assay (EMSA)--
Nucleosomes
were reconstituted with 32P-labeled or fluorescein
isothiocyanate-labeled DNA in the presence or absence of competitor DNA
by dialysis as described above. Aliquots (10 µl) of the reconstituted nucleosomes were suspended in 20 µl of 16 mM Hepes (pH
7.5), 150 mM KCl, 16% (v/v) glycerol, 1.6 mM
MgCl2, 0.8 mM dithiothreitol, 0.4 mM PMSF, 1 mM EDTA, 0.8 mg/ml of bovine serum
albumin, 0.06 mg/ml of poly(dI-dC), and 0.01% Nonidet P-40, and mixed
with 5 µl of 30 (v/v) glycerol dye. These mixtures were
electrophoresed on 4% polyacrylamide gels in 40 mM Tris
acetate, 1 mM EDTA, and 5% glycerol.
Hydroxyl Radical Footprinting--
Nucleosomes reconstituted
with the end-labeled DNA fragments corresponding to the
60 to +85
nucleosome phase were cleaved with the hydroxyl radical as described
(32, 33). Briefly, the footprinting reaction was initiated by adding an
Fe(II) EDTA solution (10 µl), which was prepared immediately before
use by mixing equal volumes of freshly prepared 125 mM
Fe(II) and 250 mM EDTA, and 28 mM sodium
ascorbate (10 µl) and 0.84% hydrogen peroxide (10 µl) on the inner
wall of the tube containing the reconstituted nucleosomes. The reaction
was allowed to run for 2 min and then quenched by adding 0.1 M thiourea (10 µl), 0.2 M EDTA (2 µl), tRNA
(10 µg), and 0.5% SDS (30 µl). The DNA was extracted with phenol,
recovered by ethanol precipitation, and resolved on 8% denaturing
polyacrylamide gels.
DNase I Footprinting--
Nucleosomes reconstituted with
end-labeled DNA fragments corresponding to the
60 to +85 nucleosome
phase were cleaved with DNase I as described previously (29). After
purification, DNA was resolved on a 6% polyacrylamide, 7 M
urea gel under denaturing conditions.
Ligation-mediated PCR (LM-PCR)--
LM-PCRs were carried out as
described (34). Briefly, HeLa cells were transfected with or without
the plasmids containing NF-E2 subunits, p18 or p45 (29), and cultured
in minimum Eagle's medium supplemented with 10% fetal bovine serum in
a humidified incubator under a 5% CO2 atmosphere for
24 h. Cells were treated with 1% formaldehyde for 10 min at room
temperature to cross-link proteins and DNA, rinsed with PBS twice, and
then incubated with 0.2% dimethyl sulfate at 20 °C for 10 min. The
reaction was stopped by addition of 1.5 M sodium acetate
(pH 7.0) and 1 M mercaptoethanol followed by washing with
PBS twice. The cells were then suspended in 1% SDS and 0.1 M NaHCO3 and heated at 65 °C for 4 h to
untie the cross-links. After preparation of the genomic DNA, piperidine was added at 90 °C for 30 min to ensure strand breakage. DNA was then purified and used for first strand synthesis extending up to the
cleaved sites using the primer (5'-TTACAAGCTCAGCTCCCTCTATCC-3'). The
synthesized DNA was ligated to the double-stranded linker oligonucleotides (5'-GAATTCAGATC-3' and
5'-GCGGTGACCCGGGAGATCTGAATTC-3') and amplified by PCR. The PCR primers
were 5'-TCCCTCTATCCCTTCCAGCATCC-3' and 5'-GCGGTGACCCGGGAGATCTGAATTC-3'.
The conditions for PCR were 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min, followed by a 10-min extension. Linear amplification of the
digested fragment was carried out by PCR primer extension with a
32P-end-labeled primer
(5'-CCAGCATCCTCATCTCTGATTAAATAAGC-3':
11,043 to
11,015 relative to
the cap site of the
-globin gene), and the amplified fragments were
resolved on 8% denaturing polyacrylamide gels.
Chromatin Immunoprecipitation Assay (ChIP Assay)--
The assay
followed a procedure published previously (35, 36). K562 cells were
cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum
in a humidified incubator under a 5% CO2 atmosphere. After
the introduction of plasmids, HeLa cells were cultured as mentioned
above. Cells were then treated with 1% formaldehyde for 10 min at room
temperature to cross-link proteins and DNA before being rinsed with PBS
twice. The cells were lysed by adding 150 µl of lysis buffer (25 mM Tris-HCl (pH 8.0), 140 mM NaCl, 1% Triton
X-100, 0.1% SDS, 3 mM EDTA, and 1 mM PMSF) and
were allowed to incubate on ice for 10 min. Sonication was followed by
centrifugation, and the supernatants containing soluble chromatin were
collected after digestion with or without MNase. The chromatin solution
was cleared with salmon sperm DNA and protein A-Sepharose for 2 h
before overnight incubation with 10 µg of anti-NF-E2 (p45) polyclonal
antibody (Santa Cruz Biotechnology). Fifty microliters of 50% protein
A-Sepharose was added to the samples and incubation carried out at
4 °C for 2 h followed by centrifugation. The pellets were
washed three times, once in 150 µl of wash buffer 1 (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS,
1% Triton X-100, and 2 mM EDTA), once with 150 µl of
wash buffer 2 (20 mM Tris-HCl (pH 8.0), 500 mM
NaCl, 0.1% SDS, 1% Triton X-100, and 2 mM EDTA), and once
in 150 µl of wash buffer 3 (10 mM Tris-HCl (pH 8.0), 0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, and 1 mM EDTA). Each wash was performed at 4 °C for 10 min.
Next, the samples were washed 3 times with 200 µl of TE buffer. The immunocomplexes were then eluted off with 1% SDS and 0.1 M
NaHCO3 and heated at 65 °C for 4 h to reverse the
cross-links. The cross-links of DNA input samples were reversed in a
similar manner. DNA from the samples was purified and subjected to PCR.
The primers 5'-GGTCAGTGCCCCACCCCCGCCTT-3' and
5'-AGATAGGAGTCATCACTCTAGG-3' were used for amplification of a 150-bp
fragment of HS2 and 5'-AGGATAGACATGCAGAAATGCATTTTAAAAATC-3' and
5'-GTTAGGGCGCACATGGGTGTTCATGCCTTCC-3' for that of a 197-bp fragment
(positions
975 to
779) of the human estrogen receptor
(ER
)
gene (37). PCR products were resolved on 2% agarose gel containing
ethidium bromide.
 |
RESULTS |
Translational Phase at HS2--
The HS2 region is very important
for
-globin gene expression and contains many transcription factor
binding sites, NF-E2 and GATA-1, for example (Fig.
1A). The binding of these
factors to their cognate motifs is critical in regulating the gene
expression, suggesting that it is crucial to investigate the status of
these cis-elements around the HS2 region. We have previously
reported (28) that there were two major nucleosome phases at HS2
in vivo. One is the
60 to +85 phase which has a nucleosome
dyad axis at its center, where the tandem MAREs are located, and the
other is the
100 to +45 phase. We have also reported that the
translational phase of the nucleosome containing the tandem MAREs and
the enhancer activity was modulated by the curved DNA located at a
distance of two nucleosomes from HS2 (29). First, we analyzed the
translational phase of the nucleosomes at HS2 when the effect from the
key nucleosome was absent. As shown in Fig. 1A, we used two
end-labeled DNA fragments corresponding to the
100 to +45 and
60 to
+85 nucleosome phases, respectively, for reconstitution of the
nucleosomes in vitro. After their reconstitution, the
nucleosomes were treated with MNase and then resolved on 4% native
polyacrylamide gel. Interestingly, the band of the nucleosome
containing the 5'-end-labeled DNA fragment corresponding to the
100
to +45 nucleosome phase disappeared immediately after MNase digestion,
whereas the bands containing the other DNA fragments remained
undigested, indicating that the 5'-region of the DNA fragment
corresponding to the
100 to +45 nucleosome phase did not bind to
histone octamers but was free because this region was not protected
from MNase digestion (Fig. 1B). These results suggest that
the nucleosome over the HS2 region bore a propensity to show a specific
position. To determine the exact translational phase, the ~146-bp
core DNA fragments were recovered after MNase digestion from the
nucleosomes reconstituted in vitro using a 200-bp DNA
fragment containing whole regions of the
100 to +45 and
60 to +85
phases and were analyzed by restriction digestion. As shown in Fig.
1C, Sau96I did not create any digested bands,
whereas BspHI and BfaI produced mainly two bands
of ~70 and 120 bp, respectively, suggesting that the major translational phases were
60 to +85. In BspHI or
BfaI digests, some minor bands were observed, indicating
that several minor phases were also present in this region as we
reported previously (28). The position shown here in the nucleosome
reconstituted in vitro was almost identical to the
60 to
+85 phase observed as in a repressive state in HeLa cells. The results
are summarized in Fig. 1D.

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Fig. 1.
Translational phases of the nucleosome
reconstituted in vitro using the HS2 fragment.
A, map of the region around HS2 of the human -LCR.
DNA sequences recognized by NF-E2, HS2NF5, and GATA-1 are
boxed. Ovals indicate the nucleosomal phases
reported previously (28). The positions relative to the -globin gene
cap site are indicated on the top. B, MNase
digestion assay of the nucleosomes (Nuc) reconstituted
in vitro using the end-labeled DNA fragments containing the
100 to +45 and 60 to +85 phases, respectively.
C, Southern blot analysis of the in vitro
reconstituted nucleosome at HS2. D, summary of translational
phases at HS2.
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Characterization of the Nucleosome Structure at
HS2--
Previously, we reported that DNA bend sites around
B-17
and
B-16 showed nucleosome positioning activity with almost unique phases, respectively (28). The positions of the neighboring nucleosomes
were abolished after removal of these sites (29). Here the HS2 region
also showed several preferential nucleosome positions (Fig. 1),
although typical curved DNA was not found by circular permutation assay
(28). In contrast, TRIF 1.00 computer software (31) predicted
significant DNA curvatures not only in the regions containing
B-17
and
B-16 but also at HS2, although the HS2 region showed
right-handed superhelicity, whereas both
B-17 and
B-16 showed
left-handed superhelicity (middle in Fig. 2). The B, NF30, and E subregions in
B-17, HS2, and
B-16, respectively, contained curved DNA. When we
performed a competition assay (bottom in Fig. 2), the
oligonucleotides containing these subregions did not compete strongly
in nucleosome formation, whereas those from the immediate vicinity (C
in
B-17, B and C in HS2, and D in
B-16) showed the strongest
competition in each region. These results suggest that the NF30
fragment in the HS2 region that had curved DNA with right-handed
superhelicity bound to the histone octamer less tightly than the other
subregions.

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Fig. 2.
DNA structure and affinity for the histone
octamer of the fragments in B-17, HS2, and B-16. Upper
panel, the nucleosome phases around HS2 of the human -LCR. The
phases are indicated by the circles as reported before (28).
The open and solid boxes show the sites of curved
DNA determined by circular permutation assay and the bend centers,
respectively (28). The probes and the
oligonucleotides are the DNA fragments and the
oligonucleotides used for the nucleosome reconstitution and for the
competition assay as competitors, respectively. The probes used are as
follows: B-17, 11,498 to 11,250; HS2,
10,989 to 10,769; B-16, 10,609 to 10,408. The
positions of the oligonucleotides are as follows: for B-17:
A, 11,369 to 11, 340; B, 11,339 to
11,310; C, 11,309 to 11,280; D, 11,279 to
11,250. For HS2: A, 10,918 to 10,889;
B, 10,888 to 10,859; NF30, 10,858 to 10,829;
C, 10,828 to 10,799; D, 10,798 to 10,769.
For B-16: A, 10,597 to 10,568; B, 10,567
to 10,538; C, 10,537 to 10,508; D, 10,507
to 10,478; E, 10,477 to 10,448; and F,
10,447 to 10,418. AT, the control oligonucleotides,
(dA)30 and (dT)30. Middle panel, the
results of curvature analysis for B- 17 and B-16 and HS2 using
TRIF software. Bottom panel, the results of EMSA using the
nucleosomes reconstituted in vitro in the presence of
competitors. The positions of the mononucleosome and the free DNA probe
are indicated as Nuc and Probe,
respectively.
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We next examined the accessibility to MNase of these fragments in the
nucleosome (Fig. 3). We subcloned HS2
fragments, A-D and NF30, which had been used as competitors (Fig. 2),
into pBluescript SK+ and reconstituted nucleosomes in vitro
using the 305-bp fragments from these subclones as probes. As shown in
Fig. 3A, all constructs, including the DNA fragment without
an insert, showed similar band intensities at the mononucleosome
position, indicating that there was little, if any, difference in
nucleosome formation among these constructs. However, when the
nucleosomes reconstituted in vitro were digested with MNase
and the ~146-bp core DNA fragments recovered were analyzed on a 6%
polyacrylamide gel, only the nucleosome containing the NF30 fragment
showed little recovery of the core DNA (shown by an arrowhead in Fig.
3B), which was even less than that from the nucleosome with
a pBluescript fragment. Because the DNA fragments used in nucleosome
reconstitution in this experiment had the same DNA sequence except for
the 30-bp insert regions and showed nucleosome formation at the same
level, the difference in MNase sensitivity should be attributable to
the insert. These results suggest that the NF-30 fragment containing
the tandem MAREs was more accessible to MNase on the nucleosome and did
not bind to the histone octamer tightly.

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Fig. 3.
Sensitivity of cloned DNA fragments from the
HS2 region to MNase after nucleosome formation. A, EMSA
of the nucleosomes. B, sensitivity to MNase. The ~146-bp
core fragments generated after MNase digestion are indicated by
arrows. pBSK, DNA fragments amplified from pBluescript SK.
The positions of the subcloned inserts are: A, 10,918 to
10,889; B, 10,888 to 10,859; NF30, 10,858 to
10,829; C, 10,828 to 10,799; D, 10,798 to
10,769. Mononuc, mononucleosome.
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Rotational Phase at HS2--
To confirm whether the NF30 region
has a similar structure on the nucleosome at HS2, we first determined
the rotational phase of the nucleosome reconstituted with the DNA
fragment corresponding to the
60 to +85 phase. Fig.
4 shows the results of hydroxyl radical
footprinting, where a 10-bp ladder pattern characteristic of the
rotational phases of nucleosomes was observed. In the NF30 region, some
of the nucleotides at each NF-E2-binding site showed accessibility to
hydroxyl radicals, indicating that a part of the NF-E2-binding site
faces outside of the histone octamer. A simple calculation based on
hydroxyl radical footprinting results (Fig. 4) predicted that DNA
wrapped around the histone core had an average number of 10.2 bp per
helical turn, which agreed with the corresponding number from the
nucleosomes in general (38). Next, we performed DNase I footprinting to
analyze the accessibility to nuclease of the tandem MAREs on the
nucleosome. The periodic 10-bp cleavage sites shown as open
triangles in Fig. 5 were observed, and many other cleaved sites (closed circle in Fig. 5), in
particular on the antisense strand, were present within the tandem
MAREs. The existence of such nuclease-sensitive sites agreed with the results in Fig. 3. These findings suggest that the tandem MAREs on the
nucleosome at HS2 are acceptable for the binding of NF- E2 by
facilitating interaction with NF-E2.

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Fig. 4.
Hydroxyl radical footprinting of the
nucleosomes containing the 60 to +85 nucleosome phase. The
densitometric scanning patterns were visualized with ImageQuant.
Square brackets indicate the tandem MAREs, and closed
circles indicate the nucleotides showing the highest sensitivity
to the radical within each of the tandem MAREs.
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Fig. 5.
DNase I footprinting analysis of the
nucleosome containing the 60 to +85 nucleosome phase.
Vertical bars and square brackets indicate the
tandem MAREs, and closed circles indicate the nucleotides
showing high sensitivity to DNase I within the tandem MAREs. Open
triangles show the periodic 10-bp cleavage sites indicating
rotational nucleosome phases.
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NF-E2 Binding to Its Cognate Motifs on the Nucleosome in
Vivo--
To confirm that NF-E2 can bind to its cognate motifs while
it is located on the nucleosome, we performed a DMS LM-PCR using non-erythroid HeLa cells. We have already determined nucleosome positions around the HS2 region in active and inactive states using
K562 and HeLa cells, respectively (28). HeLa cells do not express the
-like globin genes and exhibit a packed chromatin structure at this
locus as revealed by the absence of the erythroid-specific DNase
I-hypersensitive sites. The translational phase at HS2 in HeLa cells
mainly showed the
60 to +85 phase which is the same as observed in
the nucleosome reconstituted in vitro (Fig. 1). After
introducing NF-E2 expression plasmids in HeLa cells, the binding of
NF-E2 to its cognate motif on the nucleosome in vivo was
analyzed by genomic DMS footprinting followed by LM-PCR. Gel patterns
of the antisense strand of HS2 after LM-PCR with DNA from the
NF-E2-expressing or non-expressing cells are shown in Fig.
6. Note that the antisense strand
exhibits more prominent footprinting patterns than the sense strand
(39). The positions of guanine nucleotides, the targets of DMS
modification, matched very well those of the cleaved bands except for
the region at the tandem MAREs in cells expressing NF-E2. The region
showed significant protection against methylation. Ikuta and Kan (40) and Reddy and Shen (41) reported that the binding of NF-E2 to its motif
generated hyper-reactive patterns of guanine nucleotides within and
flanking the tandem MAREs in K562 cells. Previously, we also reported
that inducing NF-E2 expression in HeLa cells generated an increased
DNase I sensitivity at the MAREs perhaps corresponding to the changes
in the chromatin structure (29). As shown in Fig. 6, we also observed
hyper-reactive patterns of guanine nucleotides within and flanking the
tandem MAREs in HeLa cells when NF-E2 expression was induced (indicated
as closed circles). These results suggest that NF-E2 can
interact with its binding motifs on the nucleosome structure in HeLa
cells.

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Fig. 6.
Binding of exogenous NF-E2 to the tandem
MAREs in HeLa cells revealed by LM-PCR. The samples from HeLa
cells expressing (+) or not expressing ( ) NF-E2 were analyzed by
LM-PCR. The densitometric patterns were visualized with ImageQuant.
Vertical bars and square brackets indicate the
tandem MAREs. Open and closed circles indicate
the guanine nucleotides showing lower or higher intensity when NF-E2 is
expressed, respectively. Arrowheads indicate the guanine
nucleotides showing the same intensity in the presence and absence of
NF-E2.
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To confirm the ability of NF-E2 to bind the nucleosome, we next
performed a ChIP assay. After the expression of NF-E2 in HeLa cells,
protein-DNA cross-linking was performed by incubating the sample with
formaldehyde, and the complexes that formed were fragmented by
sonication. The NF-E2-containing complexes were recovered by immunoprecipitation using anti-p45 antibody, and the DNA fragments that
bound with NF-E2 were prepared after release from the fixation. The HS2
region was then amplified by PCR using the recovered DNA fragments as
templates to identify whether the HS2 region was included in these
fragments or not. A region from the ER
gene promoter, which lacks
MAREs, was used as a negative control. Based on our previous study on
the nucleosome structure in this region (42), PCR was designed to
amplify the region within a nucleosome phase. As shown in Fig.
7A, the HS2 region was
amplified from K562 cells, which express endogenous NF-E2, and also
recovered from the HeLa cells expressing NF-E2. Apparently, no bands
were amplified from the original HeLa cells, which do not express
NF-E2. We then confirmed that the recovered HS2 region from HeLa cells was derived from the nucleosome. When the cell lysate was digested with
MNase after cross-linking, the protein-DNA complexes became as small as
mononucleosomes suggesting that most linker regions were digested,
whereas nucleosome regions were retained under these conditions (data
not shown). When these samples were used for ChIP assay (Fig.
7B), the HS2 region was amplified by PCR even after
digestion with MNase, and the quantity of the amplified band differed
little between the samples with or without digestion. The control
fragment from the ER
gene, on the other hand, was not amplified from
the samples except for the input because of the absence of MAREs. This
indicates that the recovered HS2 fragments were derived from the
nucleosome. These results revealed that NF-E2 can bind to its cognate
motifs even if the binding site is located on the nucleosome.

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|
Fig. 7.
Binding of exogenous NF-E2 to the tandem
MAREs on the chromatin revealed by ChIP assay. A, samples
were prepared from HeLa cells expressing (+) or not expressing ( )
NF-E2 and from K562 cells. Templates for PCR are the input chromatin
(Input) and the DNA precipitated with (+Ab) or
without ( Ab) anti-NF-E2 antibody. B, ChIP assay
with MNase digestion. Arrowheads indicate the positions of
the PCR products of HS2. The human ER gene was used as a
control.
|
|
 |
DISCUSSION |
What Happens When NF-E2 Binds to the MAREs?--
The central role
of the human
-LCR is to regulate and enhance
-globin gene
expression in a tissue- and stage-specific manner and is mediated by
specific transcription factors (43). Two mechanisms were proposed for
the activation of globin gene expression. In the dominant chromatin
opening model, regulation of the expression of individual genes is
autonomous and is dependent on the state of transcription factors
during development (44). In this model, the LCR is simply responsible
for creating a decondensed and favorable chromatin structure in which
the globin gene promoters can interact with stage-specific
transcription factors. In the mutual interaction model, on the other
hand, the LCR is physically in contact with the individual promoters
and activates them sequentially, switching expression of the genes in
response to stage-specific factors (45, 46). The transcription factors
that bind to LCR HSs and globin promoters can homo- and heterodimerize
(47-49) and can interact with TAFII130 or cAMP-response
element-binding protein/p300, components of the transcriptional
machinery (50-53). Some transcription factors function by altering the
chromatin structure. Erythroid Krüppel-like factor binds to
SWI/SNF remodeling complex after acetylation and induces adult-type
-globin gene expression (54). NF-E2 is also involved in the
ATP-dependent nucleosome remodeling process (22, 23, 28).
NF-E2 is one of the most important factors for regulating
-globin
gene expression and consists of p45 and p18 subunits (7, 8). The p18
subunit, MafK, also heterodimerizes with Bach1 (55), Nrf1 (56), and
Nrf2 (57). Interestingly, the Bach1 heterodimer binds to MAREs
located in different regulatory elements within the human
-LCR and
creates a large looped DNA structure (20), suggesting that this
structure is involved in opening chromatin. Recently, it was reported
that the chromatin-opening function of the LCR may not be the primary
activity in the endogenous mouse or human globin locus (26, 27). The
relationship among transcriptional activation, an open chromatin
structure, and DNase I-hypersensitive sites has not been completely
elucidated, and further study is needed. Nevertheless, it is clear that
the binding of NF-E2 to the MAREs at HS of the
-LCR is critical for
high level
-globin gene expression.
Levings and Bungert (58) reported that the transcriptional activation
process of the
-globin locus is conceptually divided into four steps
as follows: (a) generation of a highly accessible LCR
holocomplex; (b) recruitment of transcription and
chromatin-modifying complexes to the LCR; (c) establishment
of chromatin domains permissive for transcription; and (d)
transfer of transcription complexes to the globin gene promoters.
Forsberg et al. (36) demonstrated that NF-E2 binds directly
in vivo to the tandem MAREs at HS2 of the human
-LCR in
K562 cells. Previously, we also reported that NF-E2 binds to the tandem
MAREs at HS2 of the human
-LCR in vitro and remodels the
reconstituted nucleosome containing the tandem MAREs at HS2 in the
presence of remodeling factors and ATP (28). From these results, we
speculate that NF-E2 first binds directly to the tandem MAREs at HS2 on
the chromatin structure, then remodels the chromatin structure in the
presence of remodeling factors and ATP, and enhances the globin gene
expression. The first step in the binding of NF-E2 to the nucleosome
may be rate-limiting and critical for the activation of globin gene
expression. Therefore, in this report we focused on this first step and
examined whether NF-E2 can bind to the tandem MAREs at HS2 on the chromatin.
How Does NF-E2 Bind to the MAREs?--
Previously, we found that
the nucleosome over HS2 was aligned by a key nucleosome located at a
distance of two nucleosomes from HS2, and the enhancer activity can be
modulated by changing the distance between the nucleosome over HS2 and
the key nucleosome (29). The key nucleosome was marked by curved DNA.
These findings suggest that the nucleosome over HS2 is crucial for the
enhancer activity.
The nucleosome reconstituted in vitro using only the HS2
fragment showed preferred nucleosome positions, which corresponded mostly to the
60 to +85 phase (Fig. 1). The
60 to +85 phase had a
nucleosome dyad axis and tandem MAREs at its center, which was an
appropriate position for NF-E2 to bind (29). The advantage of aligning
nucleosomes by the key nucleosome may be to reduce deviation in the
nucleosome phase at HS2 and to facilitate the interaction of NF-E2 with
the MAREs by adjusting the site of interaction at the dyad axis. If a
motif is well exposed to an incoming factor because it faces a specific
direction as a result of specific nucleosome positioning, protein
accessibility should be greatly improved (59). This idea has already
been demonstrated in part in a nucleosome containing a thyroid
hormone-responsive element (60).
The DNA fragment at HS2 showed an affinity to the histone octamer as
high as the DNA bend sites,
B-16 and
B-17, although the HS2
fragment did not show nucleosome positioning activity like the curved
DNAs (28). All the DNA fragments, HS2,
B-16, and
B-17, showed
almost the same structural characteristics where the sequences adjacent
to the 30-mer curved DNA but not the curved DNA sequences themselves
had the highest affinity to the histone octamer. The major difference
was that HS2 showed a right-handed superhelicity and could not
determine nucleosome positions alone, whereas the DNA bend sites,
B-16 and
B-17, showed left-handed superhelicity and could
determine unique nucleosome positions (Fig. 2). It was reported that
right-handed DNA was difficult to incorporate into nucleosomes (61),
and that is why the HS2 region did not show a fixed nucleosome position
even though it showed a higher affinity to the histone octamer.
Moreover, the tandem MAREs showed accessibility to MNase and DNase I on
the nucleosome, suggesting that the proteins can interact with the tandem MAREs on the nucleosome (Figs. 3-5). A simple calculation based
on hydroxyl radical footprinting (Fig. 4) predicted that the DNA
wrapped around the histone core of the in vitro
reconstituted nucleosome with HS2 had an average number of base pairs
per helical turn of 10.2, which agreed with the corresponding number
for the nucleosomes in general (38). This suggests that the nucleosome at HS2 is structurally the same as general nucleosomes. Although the
central three turns at the dyad axis in the nucleosome have a greater
helical density (10.7 bp per turn) than the rest of the nucleosomes, we
could not identify a difference in structure at HS2 in Fig. 4. We are
currently investigating the structure of the nucleosome over HS2 in
more detail.
Increased sensitivity to DNase I at the tandem MAREs on the HS2
nucleosome was observed (Fig. 5), which seemed to be related to the
formation of DNase I HSs. However, it was reported that the
chromatin-opening function of the LCR is not the primary activity in
the endogenous mouse or human globin locus (26, 27). The higher
sensitivity to DNase I observed here may be related to the globin gene
expression because the tandem MAREs at HS2 are critical for the gene
expression, and the alteration to the DNase I sensitivity in this
region should be caused by the modification of the nucleosome
structure. However, the formation of a core DNase I HS structure
requires binding sites for multiple factors including GATA-1, NF-E2,
EKLF, and Sp1 (62). To elucidate the relationship between the change in
DNase I sensitivity observed here and HSs, further analysis is required.
NF-E2 Binds to the MAREs at HS2 on the Chromatin
Structure--
NF-E2 can bind directly to the tandem MAREs at HS2 of
the human
-LCR in K562 cells as revealed by genomic footprinting
(39-41) and ChIP assay (36). However, it is not clear whether NF-E2 can bind to the tandem MAREs while they are located on the chromatin. There are three factors that regulate chromatin remodeling and transcription, transcription regulators, histone
acetyltransferase complexes, and ATP-dependent remodeling
complexes, which can act in different order (63). In the process of
remodeling, a sequence-specific DNA-binding protein should interact
with a binding site on the nucleosome structure where transcription is
inactive and recruit chromatin remodeling complexes such as SWI/SNF
(64). As we mentioned above, NF-E2 is involved in the
ATP-dependent nucleosome remodeling process at HS2 (22, 23,
28). Moreover, it was shown that direct physical interaction between
NF-E2 and CBP/p300 is critical for recruitment of the coactivator
complex at HS2 (65). Because chromatin remodeling is completed in K562
cells, these previous observations may just indicate that NF-E2 binds
to the MAREs on the open chromatin structure the same as naked DNA.
Here, we demonstrated the direct binding of NF-E2 on the chromatin in
HeLa cells where no NF-E2 or globin genes were expressed, and the HS2
region showed a packed chromatin structure (28), by genomic
footprinting (Fig. 6) and ChIP assay (Fig. 7). We previously tried in
vain to detect the binding using nucleosomes reconstituted in
vitro (28), suggesting that the complex of nucleosome with NF-E2
is not stable and may require proteins. It is also quite intriguing to
know how the chromatin structure is affected in NF-E2 null cells
because NF-E2 was shown to be essential for globin gene expression in
an NF-E2 null erythroleukemia cell line, CB3 (66).
 |
ACKNOWLEDGEMENTS |
We are grateful to Y. Wada-Kiyama and
T. Torigoe for helpful discussions and comments on the
manuscript. We also thank M. Yamamoto for p45 and p18 cDNA.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-298-61-6189;
Fax: 81-298-61-6190; E-mail: y-onishi@aist.go.jp.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M209612200
 |
ABBREVIATIONS |
The abbreviations used are:
-LCR,
-globin
locus control region;
HS, DNase I-hypersensitive site;
MARE, Maf-responsive element;
MNase, micrococcal nuclease;
PMSF, phenylmethylsulfonyl fluoride;
EMSA, electrophoretic mobility shift
assay;
LM-PCR, ligation-mediated PCR;
PBS, phosphate-buffered saline;
ChIP, chromatin immunoprecipitation;
ER
, estrogen receptor
;
DMS, dimethyl sulfate.
 |
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