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Biochimica et Biophysica Acta 1679 (2004) 201–213
Chromatin structure at the flanking regions of the human beta-globin locus
control region DNase I hypersensitive site-2: proposed nucleosome
positioning by DNA-binding proteins including GATA-1
Neil Davies, John Freebody, Vincent Murray*
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney NSW 2052, Australia
Received 7 January 2004; received in revised form 6 April 2004; accepted 8 April 2004
Available online 10 May 2004
Abstract
The human beta-globin locus control region DNase I hypersensitive site-2 (LCR HS-2) is erythroid-specific and is located 10.9 kb
upstream of the epsilon-globin gene. Most studies have only examined the core region of HS-2. However, previous studies in this laboratory
indicate that positioned nucleosomes are present at the 5V- and 3V-flanking regions of HS-2. In addition, footprints were observed that
indicated the involvement of DNA-binding proteins in positioning the nucleosome cores. A consensus GATA-1 site exists in the region of the
3V-footprint. In this study, using an electrophoretic mobility shift assay (EMSA) and DNase I footprinting, we confirmed that GATA-1 binds
in vitro at the 3V-end of HS-2. An additional GATA-1 site was found to bind GATA-1 in vitro at a site positioned 40 bp upstream. At the 5V-end of HS-2, DNase I footprinting revealed a series of footprints showing a marked correlation with the in vivo footprints. EMSA indicated
the presence of several erythroid-specific complexes in this region including GATA-1 binding. Sequence alignment for 12 mammalian
species in HS-2 confirmed that the highest conservation to be in the HS-2 core. However, a second level of conservation extends from the
core to the sites of the proposed positioning proteins at the HS-2 flanking regions, before declining rapidly. This indicates the importance of
the HS-2 flanking regions and supports the proposal of nucleosome positioning proteins in these regions.
Crown Copyright D 2004 Published by Elsevier B.V. All rights reserved.
Keywords: Gene expression; Nucleosome; Phylogenetic footprinting; GATA-1
1. Introduction
The human beta-globin locus contains five genes that are
expressed in a developmental stage-specific manner that
reflects their order in the beta-globin array [1]. The regula-
tion of expression is mainly at the transcriptional level and
is mediated by both proximal and distal DNA-regulatory
elements. The major distal regulatory element in the human
beta-globin locus is the locus control region (LCR) [2–4]
which is located 6-22 kb upstream of the epsilon globin
gene (Fig. 1), and consists of at least five erythroid-specific
DNase I hypersensitive sites (HS-1 to HS-5). The LCR is
required for conferring high-level globin gene expression at
all stages of erythroid development [5].
The HS-2 and HS-3 core elements of 200–300 bp
contain most of the LCR activity, and HS-2 appears to be
0167-4781/$ - see front matter. Crown Copyright D 2004 Published by Elsevier
doi:10.1016/j.bbaexp.2004.04.002
* Corresponding author. Tel.: +61-2-9385-2028; fax: +61-2-9385-
1483.
E-mail address: [email protected] (V. Murray).
necessary and sufficient for full LCR activity in transgenic
mice [6,7]. HS-2 contains multiple protein binding sites [8]
and has enhancer activity in transient expression experi-
ments [9,10]. The trans-acting factors binding in this region
include GATA-1 [11–13], NF-E2 [14] and CACC-binding
proteins [13].
DNase I hypersensitive sites (DHS) are classically
regarded as regions that are free from nucleosomes
(although this is an area of debate). This permits tran-
scription factors to bind in the region, leading to the
induction of gene expression [15]. The results of Cairns
and Murray [16] and Kim and Murray [17,18], using
bleomycin and hedamycin in vivo footprinting agents,
support the proposal that the HS-2 core region is nucle-
osome-free in erythroid cells. The results of Kim and
Murray [17,18] show evidence for positioned nucleosomes
at the 5V- and 3V-flanking regions of HS-2 in erythroid
cells in vivo. Positioned nucleosomes are often found at
the flanking regions of DHS [19–21]. The study pre-
sented here addresses the question of the identity of the
B.V. All rights reserved.
Fig. 1. Schematic of the human beta-globin gene cluster and LCR HS-2. The five expressed globin genes and their promoter regions are shown. The exploded
view of the LCR HS-2 shows ‘‘positioned’’ nucleosomes at the 5V- and 3V-boundaries, as well as the position of the putative ‘‘positioning’’ proteins. GATA-1B(the 3V-positioning protein candidate) and GATA-1A are indicated at the 3V-boundary of HS-2. The locations of transcription factor binding elements within
HS-2 are also indicated (figure adapted from Kim and Murray [18]).
N. Davies et al. / Biochimica et Biophysica Acta 1679 (2004) 201–213202
bound proteins at the 5V- and 3V-ends of HS-2. It
additionally provides information on the mechanism by
which the nucleosomes are positioned. It should be noted
that for this study, in vivo indicates that an experiment
was performed in intact cells while in vitro indicates that
cell extracts were used.
In the presence of ATP, nucleosomes can slide along
DNA [22]. Therefore, the maintenance of a nucleosome-free
HS requires a mechanism to prevent the sliding of nucleo-
somes into the site. There are two known mechanisms by
which nucleosomes can be positioned, thereby preventing
sliding. The mechanism that has been most experimentally
documented is positioning via certain DNA sequences. In
many instances, a strong correlation was found for the
dominant nucleosome positions in vivo and in vitro [23–
26]. However, results from other studies suggest the use of
caution in using in vitro nucleosome positions to predict in
vivo nucleosome positions [27].
The second mechanism by which nucleosomes can be
translationally positioned is via DNA binding proteins
which actively position or prevent the sliding of a nucleo-
some. Examples of genes in which this has been observed
include yeast STE6 and BAR1 [28], promoters in yeast
GAL1/GAL10 [29], and the promoter in Drosophila hsp 26
[30]. This is our favoured proposal for nucleosome posi-
tioning at the flanking regions of HS-2, for two reasons.
First, the results of Kim and Murray [18] show footprints in
intact erythroid cells, adjacent to the footprint deduced to be
caused by binding of a nucleosome. These footprints were
obtained using four different nitrogen mustards as damaging
agents. We propose that these adjacent footprints are caused
by transcription factors, which contribute to the establish-
ment of positioned nucleosomes in these regions. At the 3V-end of HS-2, a consensus GATA-1 site exists at the site of
the in vivo footprint, and is our candidate for nucleosome
positioning in this region. The second reason why we favour
DNA binding proteins as the major nucleosome positioning
factors at the HS-2 flanking regions is that the deduced in
vivo nucleosome footprints are found in erythroid K562
cells but not in non-erythroid HeLa cells, although the DNA
sequence is the same in both cell lines [18]. Therefore, the
DNA sequence alone cannot position the nucleosomes. The
lack of nucleosome footprints in HS-2 in HeLa cells
suggests that the nucleosomes are randomly assorted
throughout HS-2 in these cells.
GATA-1 was the first member of the GATA family of
zinc finger transcription factors to be described. It is a
haematopoietic cell-specific transcription factor that binds
to the consensus sequence (A/T)GATA(A/G) [31]. The
DNA binding domain consists of two zinc fingers of the
Cys2–Cys2 type, but generally only the C-terminal finger
and adjacent basic region are thought to be significantly
involved in DNA binding at most sites [32,33]. GATA-1
contacts DNA in both the major and minor grooves [34].
GATA-1 is essential for erythroid cell development [35–
37], and has been demonstrated to be involved in the
generation of active chromatin over almost all of the globin
and other erythroid-specific genes [38,39].
N. Davies et al. / Biochimica et Biophysica Acta 1679 (2004) 201–213 203
In this study, we investigated the proposal that nucleo-
some-positioning factors are present at the flanking regions
of HS-2. This was accomplished by in vitro methods and by
examination of the level of nucleotide conservation for HS-
2 in 12 species of mammal.
Fig. 2. DNase I footprinting analysis at the 3V-end of HS-2. A [32P]-end
labelled PCR product containing the 3V-end of HS-2 (bp 8831 to 9110) was
used for the DNase I footprinting experiments with K562 or HeLa nuclear
extracts. The sites of protection at GATA-1A, GATA-1C and GATA-1B by
K562 nuclear extract are shown. Dideoxy sequencing lanes are indicated.
2. Materials and methods
2.1. Nuclear extract preparation
K562 and HeLa cells were harvested and washed twice
with ice cold phosphate buffered saline (PBS). All subse-
quent steps were carried out on ice or at 4 jC. Cells were
split into 1.5-ml eppendorf tubes to give approximately
3� 107 cells per tube. Cells were centrifuged at 10,000
rpm for 30 s in a microfuge, the PBS was removed and the
cells resuspended in 800-Al buffer A consisting of 10 mM
HEPES, pH 7.8, 10 mM KCl, 15 mM MgCl2, 0.1 mM
EDTA, 0.1 mM EGTA, 1 mM PMSF, 10 Ag/ml aprotinin, 5
Ag/ml leupeptin and 1 mM dithiothreitol. The suspension
was incubated for 15 min before the addition of 50 Al of10% NP-40 and the suspension was vortexed for exactly 30
s. The homogenate was centrifuged at 14,000 rpm for 1 min
in a microfuge, and the supernatant discarded. Buffer C (80
Al) was added to the pellet, consisting of 0.4 M NaCl, 20
mM HEPES, pH 7.8, 7.5 mM MgCl2, 2 mM EDTA and 1
mM EGTA, 0.1 mM dithiothreitol, 0.1 mM PMSF, 10 Ag/ml
aprotinin and 5 Ag/ml leupeptin. The pellet (after checking
that the pellet was white and not yellow) was resuspended in
buffer C by flicking the eppendorf. The dithiothreitol,
PMSF, aprotinin and leupeptin were added to buffers A
and C immediately before use. The pellet was incubated in
buffer C for 20 min on a rotary shaker. The tubes were
centrifuged for 15 min, and the supernatant transferred to
another 1.5-ml eppendorf tube; 3 Al was set aside for proteinconcentration determination, and the remainder frozen in
30-Al aliquots at � 70 jC. The protein concentration was
determined using the Bradford reagent with bovine serum
albumin as a standard [40].
2.2. Oligonucleotides
The primers used to amplify the flanking regions of HS-2
for DNase I footprinting were: at the 3V-end, ND191—
ggatgcctgagacagaatgtgac, and ND193—catgccttcctcttcca-
tatcc; and at the 5V-end, ND112—ggagctgagcttgtaaaaagta-
tag and ND228—ctgagatcgtgccactgcactccag.
The following double-stranded oligonucleotide sequen-
ces were used as probes in the gel shift analyses: PCG
(Positive Control GATA-1)—cctgggtcttatcaggga [41];
GATA-1B—caaatatttatcttgcaggt; GATA-1A—tatatatttgttgt-
tatcaattgc; GATA-1C—atagaatgattagttattgt; 5VHS2—ggaa-
taagatacatgttttatt.
Competitor oligonucleotides were as follows: PCG,
GATA-1B, PCG(� )—cctgggtcttatgaggga [41], GATA-
1B(� )—caaatatttatgttgcaggt, Sp1—attcgatcggggcggggc-
gagc [42]; YY1—ctgcagtaacgccattttgcaaggcat [43].
2.3. Antibodies
Anti-GATA-1 antibodies were obtained from Santa Cruz
Biotechnology (SC-265) and Sigma (G0290). The Sigma
anti-GATA-1 antibody caused a supershift on EMSA gels,
while the Santa Cruz antibody did not produce a supershift
but inhibited formation of the GATA-1/DNA complex.
2.4. Gel mobility shift assay and DNase I footprinting
For gel mobility shift assays, oligonucleotides were
[32P]-labelled, hybridised and gel purified on a 15% (w/v)
native polyacrylamide gel. Binding reactions were carried
out at 0 jC in 20 Al of 10 mM HEPES, pH 7.8, 50 mM
potassium glutamate, 5 mM MgCl2, 1 mM EDTA, 1 mM
dithiothreitol, 5% (v/v) glycerol, and 1 Ag of poly (dI–
dC) [44]. Nuclear extract (5–10 Ag) was added to the
binding mix and incubated for 15 min. Where indicated,
1 Al of Sigma or Santa Cruz GATA-1 antibody was added
prior to the nuclear extract. [32P]-labelled purified double-
stranded oligonucleotide (100 fmol) was added and the
reaction mixtures incubated for a further 15 min. Where
N. Davies et al. / Biochimica et Biophysica Acta 1679 (2004) 201–213204
indicated, competitor oligonucleotides were added prior to
the probe at molar excess of 150- to 200-fold. The
resulting complexes were separated by electrophoresis
through 4% (w/v) polyacrylamide gels at 4 jC in 0.5�Tris–borate–EDTA buffer. Gels were dried and subjected
to autoradiography.
For DNase I footprinting, primer ND193was labelled with32P using polynucleotide kinase, and used with primer
ND191 to amplify the region from bp 8831 to 9110 contain-
ing the 3V-end of HS-2. At the 5V end of HS-2, primer ND112
was labelled with 32P and used with primer 228 to amplify the
region from bp 8247 to 8449. The resultant fragments were
purified on a 6% (w/v) native polyacrylamide gel, then used
in a DNase I footprinting reaction using the binding buffer
and procedure described above, except that the reaction was
scaled up to 30 Al. After the binding procedure described
above, 2 Al of a solution containing 2 mM CaCl2 and 2 mM
MgCl2 was added and mixed; then 0.003 to 0.12 Kunitz units
of DNase 1 (Progen) was added and incubated for 1 min at 25
jC. The reaction was stopped by the addition of 9 Al of 10%SDS and 4.5 Al of 0.1 M EDTA. Proteinase K was added (2
Fig. 3. Gel shift analysis at the proposed positioning protein site GATA-1B. The [3
14—GATA-1B. Lanes 1, 3–6—PCG. Free DNA probes (no nuclear extract) are
nuclear extract used in all other lanes. SigAb (lanes 4 and 8) indicates the additio
addition of 1 Al of Santa Cruz GATA-1 antibody. Competitor oligonucleotides (1
GATA-1B (lane 12), GATA-1B(� ) (lane 13) and Sp1 (lane 14). The GATA-1 ba
Al of 10 mg/ml) and the reaction incubated for 30 min at 37
jC. H2O (50 Al) was added, and phenol/chloroform extrac-
tion was performed followed by ethanol precipitation. The
pellet was resuspended in 7 Al of 10 mM Tris–HCl, pH 8.0,
0.1 mM EDTA, and 2 Al loaded onto a 6% denaturing
polyacrylamide gel with 2 Al of denaturing formamide dye.
Dideoxy sequencing reactions (obtained using the same
oligonucleotide primers and unlabelled PCR product) were
included on the gel. Gels were dried and subjected to
autoradiography.
2.5. HS-2 nucleotide conservation analysis
A phylogenetic footprinting analysis of the beta globin
HS-2 was performed. Sequences from 12 species of
mammal were compared using programs (Pretty Plot
and Eplotsimilarity) accessed online from the Australian
National Genome Information Service (ANGIS). The 12
species of mammal were human, chimpanzee, olive ba-
boon, cat, dog, galago, rabbit, pig, mouse, cow, goat and
rat.
2P]-labelled double-stranded oligonucleotide probes used were: Lanes 2, 7–
shown in lanes 1 and 2, HeLa nuclear extract was present in lane 6, K562
n of 1 Al of Sigma GATA-1 antibody. SCAb (lanes 5 and 9) indicates the
50-fold molar excess) were added for PCG (lane 10), PCG(� ) (lane 11),
nd and the GATA-1/Antibody supershift are shown.
N. Davies et al. / Biochimica et Biophysica Acta 1679 (2004) 201–213 205
3. Results
3.1. Analysis at the 3V-end of HS-2
Nuclear extracts were prepared from K562 and HeLa
cells and utilised in DNase I footprinting experiments and
electrophoretic gel mobility shift assays (EMSA). The
[32P]-end labelled PCR products were produced by am-
plification of the region from bp 8831 to 9110 containing
the 3V-end of HS-2 and used in the DNase I footprinting
experiments.
DNase I footprinting at the 3V-end of HS-2 (Fig. 2)
revealed protection at both consensus GATA-1 sites, GATA-
1A (8940–8945) and GATA-1B (8980–8985). These foot-
prints were obtained using a nuclear extract from erythroid
K562 cells which express the globin genes, but not with
nuclear extract from HeLa cells. A faint footprint occurred
at the GATA-1C site. These experiments demonstrate that an
erythroid-specific protein binds to the proposed NPP site
GATA-1B in vitro.
Radiolabelled double-stranded oligonucleotides were
produced containing the GATA-1B sequence and used in
gel mobility shift assays (Fig. 3). A major protein/DNA
complex (indicated as GATA-1 in Fig. 3) was observed with
the K562 nuclear extract (lane 7) which was not observed
with HeLa extract (lane 6). The addition of Sigma GATA-1
Fig. 4. Gel shift analysis of GATA-1 sites at 3V-HS-2. Double-stranded oligonucle
Lanes 6–10: GATA-1A. Lanes 11–15: GATA-1C. HeLa nuclear extract was pres
lanes. SigAb (lanes 2, 7 and 12) indicates the addition of Sigma GATA-1 antibody
(lanes 3, 8 and 13), PCG(� ) (lanes 4, 9 and 14). The GATA-1 band and the GA
antibody (lane 8) yielded a large ‘‘supershifted’’ complex
identifying the bound protein as GATA-1. The use of Santa
Cruz GATA-1 antibody (lane 9) resulted in almost complete
loss of complex formation. A 150-fold molar excess of
competitor GATA-1 oligonucleotides PCG and GATA-1B
(self) (lanes 10 and 12) resulted in loss of complex forma-
tion, while the use of competitor oligonucleotides with
mutated GATA-1 sites (11 and 13) did not. The GATA-1
sites of these latter competitor oligonucleotides were abol-
ished by replacing the G residue of the GATA motif with a
C, as this mutation is known to disrupt GATA-1 binding
[41]. The use of a competitor oligonucleotide containing an
Sp1 site (lane 14) had no significant effect on complex
formation.
Lanes 3, 4 and 5 represent EMSA using PCG, a positive
control oligonucleotide sequence which has been shown to
bind GATA-1 and is found in the EKLF gene promoter [41].
A band can be seen (lane 3) that co-migrated with the major
GATA-1B band (lane 6), and which is supershifted with
Sigma GATA-1 antibody (lane 4).
Gel mobility shift assays were conducted to investigate
the GATA-1A and GATA-1C sites. Using the K562 nuclear
extract (Fig. 4, lane 6), probe GATA-1Awas gel shifted and
gave a band that co-migrated with the GATA-1 band, but
HeLa nuclear extract (lane 10) did not produce this band.
This K562-specific complex was recognised by Sigma
otides for the 3 GATA-1 sites at 3V-HS-2 were used. Lanes 1–5: GATA-1B.
ent in lanes 5, 10 and 15 while K562 nuclear extract was used in all other
. Competitor oligonucleotides (200-fold molar excess) were added for PCG
TA-1/Antibody supershift are shown.
N. Davies et al. / Biochimica et Biophy206
GATA-1 antibody (lane 7) and gave a supershift. Loss of
complex formation was observed with addition of a 200-
fold molar excess of competitor oligonucleotide containing
a consensus GATA-1 site (PCG, lane 8) but not with a
competitor oligonucleotide with a mutated GATA-1 site
(PCG(� ), lane 9).
Probe GATA-1C was not gel shifted by an erythroid-
specific nuclear protein (lanes 11 and 15), and the minor
bands observed were not significantly affected by the
addition of Sigma GATA-1 antibody (lane 12) or com-
petitor oligonucleotides (lanes 13 and 14). This indicated
that GATA-1 binds in vitro at site GATA-1A but not
GATA-1C.
A phylogenetic footprinting analysis of the human beta
globin HS-2 was attempted. Sequences from 12 species of
mammal were investigated for this region. Fig. 5 shows a
depiction of nucleotide conservation at the 3V-end of HS-
2 (Pretty Plot). This indicated areas of sequence conser-
vation, but these regions were not conserved in all
species. Sequence conservation was also observed at the
GATA-1 sites, but again the conservation was not found
in all species.
Fig. 5. Nucleotide conservation (Pretty Plot) for the 3V-end of HS-2 GATA-1 sites.GATA-1 sites are indicated. For GATA-1A and GATA-1B, the actual GATA site
sequence alignment. The consensus sequence is indicated below the alignment and
that nucleotide. The species are arranged in decreasing order of similarity to hum
3.2. Analysis at the 5V-end of HS-2
[32P]-end labelled PCR products were amplified from the
5V-end of HS-2 (bp 8247 to 8449) and used for the DNase I
footprinting experiments (Fig. 6). Utilising K562 and HeLa
nuclear extracts, a series of footprints and enhancements
between bp 8330 and 8390 can be observed in Fig. 6. This
large footprinted region is composed of several smaller
footprints that are bordered by sites of enhancement at bps
8330, 8368, 8378 and 8390. The results are similar for K562
and HeLa nuclear extracts. However, there are several
erythroid-specific footprints including: the AGATAC site
(bp 8334–8339) that is a potential GATA-1 binding site;
and at bp 8360. These in vitro DNase I footprinting results
at 5V-HS-2 correlated with the in vivo results obtained by
Kim and Murray [17].
The erythroid-specific protein binding at the AGATAC
site (bp 8334–8339) was further investigated by gel
mobility shift analysis (Fig. 7). The AGATAC double-
stranded oligonucleotide probe is from the 8328–8349-bp
region. Two erythroid-specific bands were detected (la-
belled GATA-1 and YY1 +GATA-1) using this AGATAC
sica Acta 1679 (2004) 201–213
Sequence alignment for 12 species of mammal is shown. The three potential
is found on the other (non-coding) DNA strand, and is shown above the
a dash (in the consensus sequence) indicates that there is no consensus for
an.
Fig. 6. DNase I footprinting analysis at the 5V-end of HS-2. A [32P]-end
labelled PCR product containing the 5V-end of HS-2 (bp 8247 to 8449) was
used for the DNase I footprinting experiments with K562 or HeLa nuclear
extracts. Two footprints between bp 8330 and 8390 are shown as well as
enhancements at bps 8330, 8368, 8378 and 8390 (indicated by arrows). The
erythroid-specific footprint at the AGATAC site is indicated. The in vivo
footprint described by Kim and Murray [17] is indicated by the open
rectangle. Dideoxy sequencing lanes are indicated.
N. Davies et al. / Biochimica et Biophysica Acta 1679 (2004) 201–213 207
oligonucleotide (lane 6). Loss of complex formation was
observed at both band positions with the addition of a
200-fold molar excess of competitor PCG, containing a
consensus GATA-1 site (lane 7), but not by PCG(� ),
containing a mutated GATA-1 site (lane 8). Use of Sigma
GATA-1 antibody (lane 9) also causes loss of complex
formation at both bands. The lower band co-migrates with
a GATA-1/PCG complex (PCG, lane 3) which was super-
shifted by Sigma GATA-1 antibody (lane 4). This indi-
cated that GATA-1 protein is complexed with the
AGATAC oligonucleotide at this lower erythroid-specific
band.
The upper erythroid-specific band (YY1 +GATA-1) and
a band (YY1) were competed out by an oligonucleotide
containing a characterised YY1 site (lane 10), but not by an
oligonucleotide which does not bind YY1 (lane 11). Band
YY1 co-migrated with the characterised YY1/DNA com-
plex (lane 12). This suggested that the upper erythroid-
specific band is a complex of YY1 and GATA-1 with the
AGATAC oligonucleotide.
A phylogenetic footprinting analysis of 12 species of
mammal was also performed at the 5V-end of HS-2. Fig.
8 shows a depiction of nucleotide conservation at the 5V-endof HS-2 (Pretty Plot). This indicated areas of sequence
conservation especially between bps 8333–8341, 8353–
8366 and 8370–8387. These areas were highly correlated
with the DNase I footprints obtained in vitro (Fig. 6).
4. Discussion
4.1. GATA-1 and nucleosome positioning at the 3V-end of
HS-2
GATA-1 was found to be the dominant protein binding in
vitro at the site of the proposed nucleosome positioning
protein (GATA-1B) (Figs. 2 and 3). Therefore, the footprints
obtained at this site in vivo in erythroid cells [12,18] are
likely to be due to GATA-1 binding at this site in vivo. The
location of this footprint adjacent to a positioned nucleo-
some [18] suggests a role for GATA-1 in the maintenance of
the nucleosome position at this site. A role for GATA-1 has
recently been proposed in nucleosome positioning in the
human beta-globin intron 2 [45].
This study was initiated following the results of Kim and
Murray [17,18]. However, evidence for the in vivo binding
of GATA-1 at 3V-HS-2 is also found in an earlier study [12],
in which erythroid-specific footprints were observed at sites
GATA-1B and GATA-1A, using DMS as a footprinting
agent. Surprisingly, in view of our in vitro results (Fig. 4,
lanes 11–15), an erythroid-specific footprint was also ob-
served at GATA-1C, the non-consensus GATA-1 site. Fur-
thermore, all three in vivo footprints changed character
when the cells were treated with haemin, which is used to
induce globin gene expression [46].
Chromatin immunoprecipitation (ChIP) analysis has
been performed for GATA-1 over the entire beta-globin
cluster, a region of 75 kb, using arrays [47]. Two major sites
of in vivo GATA-1 binding were found: at the core of HS-2,
and upstream of the gamma-globin gene. Some evidence of
GATA-1 binding at the 3V-end of HS-2 was observed, but
not regarded as significant under the parameters of the
study. Although a powerful technique, ChIP is open to
some criticism because it requires that a specific region of
the protein of interest is accessible to the antibody being
used. It has been said that ChIP ‘‘requires well behaved
antibodies’’ [48]. GATA-1 is known to interact with itself
[49], with Friend of GATA (FOG) [50], EKLF and SP1 [51],
and may be involved in recruitment of histone acetylase
[52,53] and RNA polymerase II [54]. In addition, our
proposal suggests that GATA-1B would be in close prox-
imity to a positioned nucleosome. Any of these factors
could hinder the access of the antibody to the protein of
interest, especially as formaldehyde, used in ChIP to cross-
link the factor of interest to DNA, and cross-links protein to
protein. Horak et al. [47] used three antibody types to
attempt to circumvent this potential problem. However,
possible weaknesses of the ChIP technique are highlighted
by the fact that they did not observe additional in vivo
GATA-1 binding sites previously detected using ChIP in the
beta-globin region [55].
Fig. 7. Gel mobility shift analysis of the AGATAC site at the 5V-boundary of HS2. The labelled double-stranded oligonucleotide probes used were PCG (lanes
1, 3, 4), YY1 (lane 12) and 5VHS2 (lanes 2, 5–11). The 5VHS2 probe covers the region from bp 8328–8349 at the 5V-boundary of HS2 and contains the
AGATAC motif. The YY1 oligonucleotide contains a known high-affinity YY1 binding site. Free DNA probes are shown in lanes 1 and 2. HeLa nuclear
extract was in lane 5, while K562 nuclear extract was used in all other lanes. SigAb (lanes 4 and 9) indicates the addition of 1 Al of Sigma GATA-1 antibody.
Competitor oligonucleotides (200-fold molar excess) were added for PCG (lane 7), PCG(� ) (lane 8), YY1 (lane 10), and Sp1 (lane 11). The GATA-1 and YY1
bands are indicated by arrows. The band thought to represent simultaneous binding of GATA-1 and YY1 is indicated.
N. Davies et al. / Biochimica et Biophysica Acta 1679 (2004) 201–213208
It should be noted that GATA-2, which is also present in
erythroid cells, may also bind at the GATA-1B site. The use
of Santa Cruz GATA-1 antibody (Fig. 2, lane 9) results in
loss of the GATA-1 complex, but a minor band slightly
above this can be seen, which may be due to GATA-2
binding. This is very similar to results previously obtained
for GATA-2 gel migration relative to GATA-1 [56]. Why
Sigma GATA-1 antibody (Fig. 2, lane 8) does not produce
this result is unclear, though the two antibodies clearly have
a dissimilar effect on GATA-1 binding in EMSA.
GATA-1 was also shown to be the dominant protein
binding in vitro at the GATA-1A site (Fig. 4, lanes 6–10).
As stated previously, an in vivo haemin-inducible erythroid-
specific footprint has been reported at this site [12]. In
addition, the results of Kim and Murray [18] suggest a
possible in vivo footprint at this site, though the location of
the site at the extreme end of the gel makes interpretation
difficult. We can only speculate on the role, if any, of the
GATA-1A site at the 3V-end of HS-2. Although we propose
that the GATA-1 protein at the GATA-1B site is involved in
nucleosome positioning, it is possible that GATA-1B as well
as GATA-1A perform a combination of roles at the 3V-end
of HS-2. GATA-1 has been shown to perform a number of
functions, including perturbation of nucleosome binding
[57], recruitment of histone acetylase [52] including at
HS-2 [53], and recruitment of RNA polymerase II, includ-
ing at HS-2 [54]. These functions could all be considered to
be involved in activation of chromatin.
The observation that GATA-1 can disrupt a nucleosome
[57] could be linked to our proposal that GATA-1 is
positioning a nucleosome. GATA-1 is one of the earliest
markers of red cell differentiation [58] and could be in-
volved in chromatin remodelling at a very early stage, both
by recruitment of remodelling activities (e.g. histone acety-
lase), and directly (by nucleosome perturbation). If GATA-1
is directly involved in nucleosome disruption at the HS-2
region, this would indicate that the protein would then be
ideally placed to adopt the role of preventing the nucleo-
somes from sliding into the newly created HS. The proposal
that GATA-1B recruits histone acetylase is also attractive,
due to the hypothesised proximity of GATA-1B to the
positioned nucleosome.
The results of the clustal alignment for the nucleotide
sequences of 12 mammal species support our proposal for
Fig. 8. Nucleotide conservation (Pretty Plot) for 5V-HS-2. Sequence alignment for 12 species of mammal is shown. The site of the non-consensus AGATAC
site at bp 8334–8339 is highlighted. The sites of in vitro enhancement at bp 8330, 8368, 8378 and 8390 are indicated. The consensus sequence is shown below
the alignment and a dash (in the consensus sequence) indicates that there is no consensus for that nucleotide. The species are arranged in decreasing order of
similarity to human.
N. Davies et al. / Biochimica et Biophysica Acta 1679 (2004) 201–213 209
nucleosome positioning at the 3V-end of HS-2 (Fig. 9).
These alignments were generated after the predicted sites of
the nucleosome and positioning proteins were formulated
from in vivo and in vitro results, and the Eplotsimilarity
graph shows a striking correlation to the schematic outline
of our proposal (Fig. 9). It can be seen from this ‘‘similar-
ity’’ plot that there are in general three tiers of conservation.
The region of highest conservation is shown to lie within the
374-bp XbaI–HindIII fragment which is historically taken
to represent the core of HS-2 and is the minimal region
capable of conferring position independent expression of the
beta-globin gene [8,13]. There is a secondary tier of
conservation that extends from this core region to points
that are very close to the sites of the proposed positioning
proteins at the 5V and 3V ends of HS-2. After these points,
there is then a rapid decline in sequence conservation to a
‘‘background’’ level of conservation.
The sites of GATA-1A and GATA-1B are shown to lie
within the region of secondary conservation, before it
declines to background level. This is a compelling result,
which did not form any part of our original positioning
protein proposal. It can be seen that there is strong
conservation of the GATA-1B site, except for the second
‘‘A’’ of the GATA site (Fig. 5). GATA-1 has been shown to
bind to a variety of sites in addition to the consensus
GATA-1 site [44], and GATA-1 may recognise these varia-
tions in vivo.
The site of GATA-1A (Fig. 5) is less conserved, but is
conserved across the human, chimpanzee, olive baboon,
galago and rabbit. This may reflect differences that have
arisen between species over the course of evolution, and the
GATA-1A site may play a significant role in those species in
which it is found. This may also apply to the incomplete
conservation at GATA-1B. Examples of these ‘‘differential
footprints’’ and their significance can be found in the globin
genes. One example is the SSP-binding site in the gamma-
globin gene promoter [59], which is conserved in anthro-
poid primates only. This factor is implicated in the differ-
ential expression of gamma-globin and beta-globin genes
[60].
4.2. Nucleosome positioning at the 5V-end of HS-2
The footprints observed between bp 8330 and 8390 (Fig.
6) show a marked correlation to the in vivo footprinting
results previously reported [17], in which the in vivo
footprint is reported to be from 8332 to 8385. The DNase
I footprinting results are similar for K562 and HeLa nuclear
extract, however, there are some erythroid-specific foot-
prints, for example at positions 8336 and 8360. Examination
of the results of Kim [17] reveals partial footprinting in the
region 8330 to 8390 in HeLa cells, though the adjacent
positioned nucleosome is found in K562 cells only. This
suggests that a series of ubiquitous factors bind in this
Fig. 9. Summary diagram of the human beta-globin HS-2 and areas of sequence conservation. The horizontal bar indicates the XbaI/HindIII fragment taken to
represent the accepted HS-2 core. The positions of GATA-1A and GATA-1B are shown. The position of the 5V-HS-2 footprints obtained in vivo and in vitro is
indicated. Numbering is from the human beta-globin sequence (GenBank accession NG 000007). The exploded diagram shows the relation of the
Eplotsimilarity graph to known and proposed features of HS-2. The Eplotsimilarity graph was obtained using a 12-bp moving window. The diagram is
approximately to scale.
N. Davies et al. / Biochimica et Biophysica Acta 1679 (2004) 201–213210
region, but that a small number of erythroid-specific factors
are responsible for key activities in this region, including the
establishment of nucleosome positioning. Nucleosome rear-
rangement by erythroid proteins at the 5V-end of HS-2 has
been reported in an in vitro chromatin-assembled LCR
system [61].
An erythroid-specific DNase I footprint is observed at
position 8334–8339 at the 5V-boundary of HS-2 (Fig. 6). In
view of the 3V-HS-2 GATA-1 sites, it is provocative that a
(non-consensus) GATA-1 site exists at this point—AGA-
TAC. A probe containing this site suggested the binding of
GATA-1 at the lower erythroid-specific band in gel shift
analysis (Fig. 7). Evidence from both competitor oligonu-
cleotides and GATA-1 antibody indicates that GATA-1
protein is bound at this sequence. We speculate that
GATA-1 could be involved in nucleosome positioning at
the 5V-end of HS-2.
There was a second erythroid-specific (upper) band.
Surprisingly, addition of GATA-1 antibody or competitor
oligonucleotide results in loss of this complex. Competitor
oligonucleotide results tentatively suggest that this band is
due to the simultaneous or co-operative binding of YY1
and GATA-1. The site in the AGATAC oligonucleotide
thought to bind YY1 is the sequence TACATGTT, which
is similar to a known high affinity YY1 binding consensus
GACATNTT [62]. Further experiments are required to
clarify this point. Simultaneous and co-operative binding
of GATA-1 and YY1 has been previously reported, at the
core of HS-3 [63] and at the epsilon globin silencer
[64,65].
N. Davies et al. / Biochimica et Biophysica Acta 1679 (2004) 201–213 211
For the sequence alignment (Fig. 9) at the 5V-end of
HS-2, the results are similar to the 3V-end, with the
secondary tier of conservation extending to the site of
the in vitro and in vivo footprints reported [17], after
which conservation declines rapidly to background level.
The sequence alignment in this region (Fig. 8) reveals a
series of blocks of high conservation which show a
correlation to the series of sub-footprints in vitro (Fig.
6). The AGATAC site at 8334–8339 is not highly con-
served. However, it is possible that the site may play a
significant role in the human HS-2.
4.3. General discussion of HS-2 nucleosome positioning
From previous studies [12,18], it appears that occupation
of the GATA-1B site and the sites at the 5V-HS-2 is
continuous rather than transient, since continuous occupa-
tion would be required to produce the reported footprints.
This is in agreement with a nucleosome positioning protein
proposal [22] that nucleosome positioning by a DNA
binding factor requires that the factor be continuously bound
to the DNA binding site, due to the phenomenon of ATP-
dependent nucleosome movement.
The LCR HS core elements are responsible for most of
the activity of the LCR in transgenic assays [66–69]. The
question then arises as to the role of the HS-2 flanking
regions—the significance of which is supported by the
existence of the second ‘‘tier’’ of conservation which
extends out to the points of the proposed positioning
proteins (Fig. 9). There is evidence that the LCR HS
flanking regions may lead to synergistic enhancement of
expression, whereas the cores alone yield only additive
expression [70]. In addition, in beta-thalassemic mice,
therapeutic levels of beta-globin gene expression are
attained in the presence of the HS-2, -3 and -4 cores and
flanking regions, but not with the HS cores alone [71]. In
the LCR holocomplex model [66,72], the flanking sequen-
ces may help position the HS cores in a manner that aids
their interaction [70].
The results of previous in vivo studies of HS-2 in this
laboratory [16–18] conflict with the results of other
researchers [73,74], who report that nucleosomes are found
in vivo throughout the HS-2 region in both erythroid K562
cells and non-erythroid HeLa cells, and are positioned by
sites of DNA bending found 5V and 3V of the HS-2 core. We
believe the results of Kim and Murray [17,18] and Cairns
and Murray [16] to more truly represent the in vivo
situation, since they were obtained solely in intact cells.
The results of Onishi et al [73] and Onishi and Kiyama
_Hlt70162939[[74], while compelling, were in part deduced
using plasmid DNA and intact, extracted nuclei. Despite
this, it is possible that histone octamers are present along the
entirety of HS-2, but that they are modified; for example by
SWI/SNF complexes or histone acetylation. This could
explain why they do not produce in vivo footprints in the
studies of Cairns and Murray [16] and Kim and Murray
[17,18]. In this alternative proposal, the bound factors at the
flanking regions of HS-2 would then define the boundaries
of, and perhaps play a part in the creation and maintenance
of, this region of modified chromatin.
Acknowledgements
This research was supported by the National Health and
Medical Council of Australia. NPD was supported by an
Australian Postgraduate Award.
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