Download b-Globin locus control region HS2 and HS3 interact structurally and

1180±1190 Nucleic Acids Research, 2003, Vol. 31, No. 4
DOI: 10.1093/nar/gkg217
b-Globin locus control region HS2 and HS3 interact
structurally and functionally
David A. Jackson, Jennifer C. McDowell and Ann Dean*
Laboratory of Cellular and Developmental Biology, NIDDK, NIH, Bethesda, MD 20892-2715, USA
Received November 1, 2002; Revised and Accepted December 13, 2002
ABSTRACT
The overall structure of the DNase I hypersensitive
sites (HSs) that comprise the b-globin locus control
region (LCR) is highly conserved among mammals,
implying that the HSs have conserved functions.
However, it is not well understood how the LCR
HSs, either individually or collectively, activate
transcription. We analyzed the interactions of
HS2, HS3 and HS4 with the human e- and b-globin
genes in chromatinized episomes in fetal/embryonic
K562 cells. Only HS2 activates transcription of
the e-globin gene, while all three HSs activate the
b-globin gene. HS3 stimulates the b-globin gene
constitutively, but HS2 and HS4 transactivation
requires expression of the transcription factor
EKLF, which is not present in K562 cells but is
required for b-globin expression in vivo. To begin
addressing how the individual HSs may interact
with one another in a complex, we linked the
b-globin gene to both the HS2 and HS3. HS2 and
HS3 together resulted in synergistic stimulation of
b-globin transcription. Unexpectedly, mutated, inactive forms of HS2 impeded the activation of the
b-globin gene by HS3. Thus, there appear to be distinct interactions among the HSs and between the
HSs and the globin genes. These preferential, nonexclusive interactions may underlie an important
structural and functional cooperativity among the
regulatory sequences of the b-globin locus in vivo.
INTRODUCTION
The ®ve members (5¢-e, Ag, Gg, d, b-3¢) of the human b-globin
gene family are expressed sequentially during development
(1), and depend for their high level expression on the locus
control region (LCR), a complex, 15 kb regulatory element
10±60 kb upstream of the globin structural genes (2). The LCR
encompasses four highly conserved (3) subdomains,
HS1±HS4, which were originally identi®ed as DNase I
hypersensitive sites (HSs) (4,5). The LCR core sequences
and the globin gene promoters share motifs for a small number
of transcription factors (1). These include GATA-1 motifs,
Maf recognition elements (MAREs, recognized by NF-E2 and
other factors) and CACCC motifs [recognized by Kruppel-like
proteins such as erythroid Kruppel-like factor (EKLF)] (6±8).
Despite considerable effort, it remains unclear how the LCR
HSs, either individually or collectively, activate transcription
(9). When individual HSs are linked to the globin genes in
transgenic mice, they appear to have distinct globin gene
targets (10). However, there has been less consistency in
studies when individual HSs have been deleted and/or
substituted for one another in the context of the complete
human locus introduced into transgenic mice. In some cases,
core HS deletion preferentially reduced the activity of
particular globin genes (11,12). Other investigators found
that deletion of HS2, HS3 or HS4 core sequences individually
drastically lowered transcription of all the genes (13,14) and
lessened the ability of the other non-mutated sites to form their
characteristic DNase I-sensitive structures (14,15). The latter
observations are consistent with the idea that the HSs act as a
single synergistic entity, which has been termed the
holocomplex (16). Further highlighting the complexity of
LCR function, larger deletions encompassing individual HS
cores with some ¯anking sequence were only modestly
deleterious to transcription of the globin genes in either
human transgenic loci in mice or in the mouse locus itself
(17±20), and the remaining sites were able to form (21).
These observations suggest an additive interaction, with
considerable functional redundancy among the sites.
We have undertaken a reassembly approach to try to
understand how the LCR functions. We have selected
400±600 bp LCR HSs sequences, which subsume the majority
of LCR activity, encompass the characterized transcription
activator sites shared by the LCR HSs and globin promoters,
and are capable of forming their characteristic HS structure in
chromatin (reviewed in 1). To avoid potential integration
position effects while still analyzing LCR function in the
context of chromatin in vivo, we have employed nuclear
episomes. These minichromosomes are assembled into
physiological chromatin and are maintained extrachromosomally at moderate copy number in human embryonic/fetal
erythroid K562 cells. In earlier work, we studied interactions
between a 4 kb human embryonic e-globin gene and the
*To whom correspondence should be addressed at Building 50, Room 3154, 50 South Drive, MSC 8028, Bethesda, MD 20892-8028, USA.
Tel: +1 301 496 6068; Fax: +1 301 496 5239; Email: [email protected]
Present addresses:
David A. Jackson, The US Army Center for Environmental Health Research, Fort Detrick, MD, USA
Jennifer C. McDowell, NIH Intramural Sequencing Center, NHGRI, NIH, Bethesda, MD, USA
Nucleic Acids Research, 2003, Vol. 31, No. 4
human HS2 core sequence in minichromosomes. These and
other studies provided evidence that a distant enhancer and
gene promoter mutually affect each other's chromatin conformation (22±24), suggesting they participate in a shared
structure. Our aim in the present studies was to investigate
whether this property is shared by other globin gene promoters
and among the HSs themselves (e.g. in a holocomplex), as
would be expected if they interact in chromatin, whether
additively or synergistically.
Expression of the adult b-globin gene in animals requires
the erythroid transcription factor EKLF (25,26), and work
from several laboratories has implicated EKLF in HS3
function and hypersensitivity (27±30). K562 cells, which
have an embryonic/fetal phenotype, express neither EKLF nor
the adult b-globin mRNA (31), and HS3 sequences in the
natural K562 b-globin locus are very resistant to DNase I
digestion (32; A.Dean, unpublished results). These observations suggest that EKLF might be required for adult b-globin
transcription and HS3 function in K562 cells. Therefore, we
created K562 cell lines in which EKLF was stably expressed
and also identi®ed conditions for highly ef®cient expression of
EKLF in transiently transfected cells.
We found that HS2, HS3 and HS4 interact differently with
the e- and b-globin genes and differently from one another
with the same gene. HS2 transactivated both the e- and
b-globin genes although activation of the b-globin gene
required expression of the adult erythroid transcription factor
EKLF (25,26). HS3 failed to activate the e-globin gene but
strongly activated the b-globin gene in an EKLF-independent
manner. HS4 also failed to activate the e-globin gene and
stimulated b-globin transcription weakly even in the presence
of EKLF. To begin studying the interactions among the
various HSs, we inserted both HS2 and HS3 in their natural
order upstream of the b-globin gene. HS2 and HS3
synergistically stimulated b-globin transcription in the presence of EKLF. Interestingly, linking HS3 to HS2 constructs
altered its chromatin structure, and linking HS3 to HS2
mutants with impaired transactivation ability reduced transcription to levels lower than those observed with HS3 alone.
These results suggest that the linked HS sites interact with one
another in the context of the b-globin gene.
MATERIALS AND METHODS
Plasmids
Plasmids were constructed using conventional techniques
(33). Minichromosomes were constructed in p220.2 (34) or a
variant (see below). p220.2 contains the Epstein±Barr virus
(EBV) origin of replication, a transcription unit for EBNA-1
which is required for replication, and a hygromycin resistance
gene which permits selection and maintenance of the plasmid
in human cells. To facilitate cloning of b-globin sequences
into p220.2, NaeI and NotI restriction sites were introduced
into the polylinker of p220.2 creating p220Nae/Not by sitedirected mutagenesis (QuickChange kit; Stratagene). Stable
expression vectors for the transcription factor EKLF were
assembled in a variant of p220Nae/Not, p220neo, which
carries a neomycin resistance marker from pMC1neopolyA
(Stratagene) instead of the hygromycin resistance gene.
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We studied the b-globin LCR core HS sequences that span
the characterized transcription activator sites of each: HS4,
SacI±SspI (GenBank coordinates 954±1338); HS3, PstI±AvaII
(GenBank coordinates 4348±4942); HS2, HindIII±XbaI
(GenBank coordinates 8486±8860) (3). A 3.7 kb EcoRI
e-globin genomic fragment (GenBank coordinates 17482±
21233) and a 4.5 kb b-globin genomic PstI fragment
(GenBank coordinates 59855±64302) were used. GenBank
coordinates refer to locus NG_000007 (25 April 2001
version). The ±87 G®C thalassemia transversion (31) was
introduced into the b-globin promoter in pBSb using the
QuickChange kit (Stratagene), veri®ed by sequencing and
used to create the mutant form of the gene in minichromosomes. Construction of wild-type and mutant HS2 e-globin
minichromosomes has been described (35). HS2 variants
containing mutations in the NF-E2 and CACCC transcription
factor binding sites (24) were linked to the b-globin gene.
Mouse EKLF expression vectors were created by transferring EcoRI±BamHI segments of pSG5 EKLF (wild-type)
and pSG5 EKLFDPro [DNA-binding domain (DBD) only
deletion mutant] (8,36) into pCI (Promega). To create EKLFexpressing minichromosomes, the BglII±ClaI portion of pCI
EKLF and of pCI EKLFDPro were cloned into p220neo.
Noteworthy differences between the mouse and human forms
of EKLF are not apparent (37).
RNA protection assays
RNA was isolated from K562 cell clones using the PureScript
kit (Gentra Systems). Probes were synthesized and radiolabeled using the MaxiScript system (Ambion) and RNA
protection assays were performed using RPA II or RPA III kits
(Ambion). The e-globin probe protects a 149 nt band from
endogenous K562 e-globin transcripts and a novel 135 nt band
from minichromosomal transcripts (35). Templates for the
synthesis of radiolabeled probes that recognize either the 5¢ or
the 3¢ sequences of mouse EKLF were constructed in pBS
from pSG5 EKLF. The 5¢ 225 nt probe protects a fragment of
160 nt. A 154 nt band is also observed presumably due to an
A/T-rich region at the 3¢ end of the EKLF sequence. The 445 nt
3¢ probe protects a 401 nt portion of the EKLF message.
Radiolabeled b-globin probes were synthesized from the
genomic pBSbDBam template created by deleting the 3¢
portion of the b-globin gene from an internal BamHI site at
+481 to the downstream BamHI site in the pBS polylinker.
The 603 nt long probe protects a 114 nt fragment of the
b-globin message corresponding to the ®rst exon and a 204 nt
fragment spanning most of the second exon. The 229 nt short
probe protects a 189 nt segment of the second exon. Human
poly(A)+ bone marrow RNA was obtained from Stratagene for
use as a b-globin RNA positive control.
Transfections and cell culture
MEL cells were maintained in DMEM with 10% fetal bovine
serum and 100 mM glutamine. K562 cells were propagated in
RPMI 1640 with 10% fetal bovine serum, 100 mM glutamine
and 200 mg/ml hygromycin (Roche) and/or 200 mg/ml G418
(Invitrogen/Life Technologies) as appropriate. K562 cells
were collected by centrifugation, washed twice in a large
volume of RPMI 1640 with 10% fetal bovine serum and
suspended in this medium at 2±3 3 106 cells/300 ml on ice.
The cells were electroporated (38) in the presence of 10 mg of
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Nucleic Acids Research, 2003, Vol. 31, No. 4
plasmid DNA to create minichromosomal cell lines. EKLF
expression minichromosomes were super-transfected into
clonal cell lines already carrying globin minichromosomes
without dif®culty. The copy number, between 10 and 20
copies/cell, and integrity of minichromosomes in clonal cell
lines were analyzed by Southern blotting. Within this
moderate copy number range, copy number effects were not
evident. Electroporation conditions for transient transfections
were the same as above. Unless otherwise noted in the ®gure
legends, clonal minichromosome-containing cells were
transfected with 25 mg of EKLF expression plasmid or
empty vector, with pBS added to 60 mg of total DNA.
Approximately 80% of cells were transfected with pEGFP
(Stratagene) under these conditions, and dose±response
experiments demonstrated that b-globin transcription had
reached a plateau.
Nuclear extract preparation and western blotting
Nuclear extracts were prepared (39) from 1±2 3 107 cells.
Total protein yield was typically 2±3 mg/ml in a total volume of
100 ml of buffer D. EKLF expression was analyzed by SDS±
PAGE and western blotting using a Novex Mini-Cell apparatus
according to the manufacturer's recommendations. Samples of
30 mg of protein were fractionated in 8% resolving gels. The
quantity and quality of extracts was con®rmed by examining a
Coomassie Blue-stained gel run in parallel. EKLF in blotted
nuclear extracts was detected using an anti-EKLF polyclonal
antibody and the Enhanced Chemiluminescence system (ECL,
Amersham-Pharmacia).
Restriction enzyme accessibility
Nuclei of K562 cells carrying various minichromosomes were
prepared (35) and aliquots (2±5 3 107 nuclei) were incubated
with 100 U of restriction enzyme for 30 min at 37°C,
conditions previously determined to obtain maximum digestion of chromatin. After puri®cation, the DNA (20 mg/sample)
was cut to completion with a second restriction enzyme,
puri®ed, separated on 1% agarose gels and transferred to nylon
membranes by Southern blotting. The membranes were
hybridized to the 32P-labeled DNA probes described in the
text and ®gure legends, and the results quantitated using a
PhosphorImager (Molecular Dynamics) and ImageQuant
software. The percent digested was calculated by quantitating
the intensity of the cut/(cut + uncut) signals.
RESULTS
We previously showed that the chromatin structures of the
human e-globin gene promoter and a linked HS2 core
sequence were interdependent using minichromosomes stably
maintained in K562 cells. To ask whether such a structural and
functional interdependence is a general feature of HS±globin
gene promoter interaction and/or whether there are preferential interactions between particular HSs and promoters, we
constructed minichromosomes containing 4 kb genomic
fragments, from ~2 kb upstream to ~2 kb downstream of the
transcription start site, from the human e- and b-globin genes
with and without HS2, HS3 and HS4 core sequences. The
inserted globin sequences are schematically depicted in
Figure 1.
Figure 1. Structures of the b-globin and e-globin genes studied on minichromosomes. The various HS cores (HS2, 374 bp; HS3, 594 bp; HS4,
384 bp; see Materials and Methods) of the b-globin LCR were inserted
2±2.7 kb upstream of the globin transcription start sites in the natural
genomic orientation. The black line and rectangles represent globin gene
sequences and the gray line represents vector sequences.
Expression of EKLF at high levels in transfected
minichromosome cell lines
Because K562 cells do not express EKLF, which is required
for the expression of the adult b-globin gene from an intact
b-globin locus in animals (25,26), we anticipated that forced
expression of EKLF would be required for expression of
minichromosomal b-globin genes, and possibly for the
function of HS3 (27±30). We employed both stable and
transient vectors to express EKLF in K562 cells. To construct
the vector for stable EKLF expression, we replaced the
hygromycin resistance cassette in the minichromosome backbone with a neomycin resistance gene and introduced an
EKLF or control transcription unit into this neoR minichromosome. Hence, it was possible to select simultaneously for the
globin- and EKLF-expressing minichromosomes.
We devised transient transfection conditions in which up to
80% of electroporated K562 cells express green ¯uorescent
protein (Fig. 2A) and used these conditions to transfect K562
cells carrying globin minichromosomes with an expression
vector containing an EKLF expression cassette. Figure 2B
presents a western blot of nuclear extracts from similar
numbers of transiently (lane 2) and stably (lanes 3 and 4)
transfected K562 cells probed with anti-EKLF. As positive
controls, we assayed recombinant six histidine tagged EKLF
expressed in bacteria (lane 7) and nuclear extract prepared
from MEL cells (lane 5), which normally express EKLF and
the endogenous adult b-globin gene. Transfected K562 cells
express abundant EKLF compared with MEL cells. The
pattern and migration of EKLF bands from transfected K562
cells and from MEL cells was identical, suggesting that the
protein was similarly post-translationally modi®ed in both cell
types (40). Recombinant EKLF containing a six histidine tag
migrates more slowly as expected.
Effects of EKLF and HS2, HS3 and HS4 on e-globin
gene transcription
HS2 sequences situated 2 kb upstream of the e-globin gene
transcription start site in a minichromosome stimulated
transcription of the gene more than 100-fold (35). To ask
whether HS3 and HS4 could also stimulate e-globin transcription, we inserted the HS3 and HS4 core sequences (Fig. 1)
into the same position in the minichromosome and created
clonal cell lines carrying HS3e or HS4e along with either a
control or EKLF expression minichromosome.
Nucleic Acids Research, 2003, Vol. 31, No. 4
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Figure 2. EKLF is expressed at high levels in transfected cells and stable
minichromosome cell lines. (A) Fluorescence microscopy of K562 cells
transfected with pEGFP. At 36 h after transfection up to 80% of cells
expressed GFP under the conditions employed. (B) Western blot of protein
extracts (30 mg) from K562 cells transiently transfected with pSG5EKLF
and from K562 cell lines stably expressing EKLF from minichromosomes.
After PAGE and transfer to a nylon membrane, the blot was probed with
anti-EKLF polyclonal antibodies and proteins detected by ECL. Lane 1,
K562 cells transfected with pSG5 empty vector; lane 2, K562 cells transfected with pSG5EKLF; lane 3, high-expressing K562 cell clone carrying
an EKLF minichromosome; lane 4, low-expressing K562 cell clone carrying
an EKLF minichromosome; lane 5, MEL cell extract; lane 6, K562 cell
extract; lane 7, recombinant 6-His tagged EKLF (D.A.Jackson and A.Dean,
unpublished results). Mr, molecular weight markers.
RNA protection was used to determine the abundance of
e-globin and EKLF RNA. e-Globin transcripts from the
minichromosomes are marked by a mutation in the 5¢ UTR
such that RNase digestion produces shorter protected fragments from them than from transcripts of the endogenous
genes. The results for several clones carrying HS3e (Fig. 3A)
or HS4e (Fig. 3B) and either an EKLF or a control expression
minichromosome (vector) indicate that neither HS3 nor HS4
stimulates transcription of the e-globin gene even when EKLF
RNA is expressed at a high level compared with MEL cells
(see Fig. 2B). e-Globin transcription from the endogenous
locus exhibits clone to clone variability (24,35) but is independent of EKLF expression (Fig. 3A and B, endogenous).
Others (28,31,41) have observed that EKLF can transactivate
transiently transfected reporter genes in K562 cells as well as
transfected minichromosomes containing the b-globin gene
(see below). Therefore, we conclude that HS3 and HS4 are not
able to interact functionally with the e-globin gene in the
context of K562 cells and that overexpression of EKLF alone
in the K562 cellular milieu is not suf®cient to mediate
interaction. Overall, the lack of an EKLF effect on e-globin
transcription is consistent with observations made in EKLF
knockout mice (25,26).
Effects of EKLF and HS2, HS3 and HS4 on b-globin
gene transcription
Endogenous b-globin RNA is undetectable by RNA protection
analysis in K562 cells, and overexpression of EKLF does not
Figure 3. Effects of EKLF and HS2, HS3 and HS4 on e-globin gene transcription. Independent K562 cell clones were established which carried two
minichromosomes, either hygR HS3e or HS4e, plus neoR EKLF or empty
vector minichromosomes. (A) RNA was isolated from HS3e clones and
assayed by RNA protection separately for e-globin and EKLF RNA.
Representative RNA protection results are shown. Actin RNA served as the
load control. M, marker DNA ladder in nucleotides. (B) RNA protection
results for representative HS4e clones as described in (A). (C) Column
graph of e-globin expression by the HS3e and HS4e clones in the presence
(black bars) and absence (gray bars) of EKLF. Means of clones 6 SEM are
shown (n = 4±8 clones with data determined 3±5 separate times for each
clone). The results are compared to transcription by HS2e clones and clones
containing the e-globin gene (e) unlinked to a LCR HS site (24).
stimulate transcription from the chromosomal b-globin gene
(Fig. 4A, lanes 20±21) (31). Similarly, there was only trace
signal from the b-globin gene on minichromosomes in K562
cells, which was not altered by transiently transfected EKLF
(b, Fig. 4A, lanes 1±4, and B). To ask whether LCR core
sequences affected transcription from the b-globin minichromosomes, we analyzed clonal cell lines carrying the
b-globin gene linked to HS2, HS3 or HS4. We also tested the
effect of EKLF expression on b-globin transcription. RNA
isolated from the transfected cells was analyzed by RNA
protection for the abundance of b-globin and EKLF RNAs.
We studied three or four clones for each construct, and
Figure 4A depicts the results for two different clones per
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Figure 4. Effects of EKLF and HS2, HS3 and HS4 on b-globin gene transcription. K562 cells and K562 clones carrying a b-globin gene on a minichromosome were transiently transfected with pCI EKLF using conditions (see Fig. 2) which resulted in greater than 80% of cells expressing a test reporter
gene. (A) RNA was isolated and RNA protection experiments were carried out separately to measure b-globin (upper panel) and EKLF RNA (lower panel).
Actin RNA served as the load control. Representative results for two clones (A and B) for each construct are presented. ±, transfection with empty vector; +,
transfection with pCI EKLF. Lanes 1±4, b minichromosome cell lines (no HS site); lanes 5±8, HS2b minichromosome cell lines; lanes 9±12, HS2b minichromosome cell lines with the promoter ±87 thalassemia mutation; lanes 13 and 14, untransfected HS3b minichromosome cell lines (transfected HS3b cell
lines are not shown on this gel); lanes 15±18, HS4b minichromosome cell line; lane 19, MEL cell RNA control; lanes 20 and 21, parent K562 cells. Controls
fo b-globin RNA include HS3b (lane 22) and human bone marrow RNA (lane 23). M, DNA markers in nucleotides. (B) Column graph depicting the
abundance of b-globin RNA (6 SEM) measured by RNA protection in K562 cells carrying various minichromosomes, either transiently transfected with
empty vector (gray bars) or with pCI EKLF (black bars). b-Globin RNA is normalized for loading and minichromosome copy number.
construct. Multiple experiments are graphically summarized
in Figure 4B.
Linking the b-globin gene to HS2 renders the gene
responsive to EKLF, with a 4.4-fold increase in b-globin
RNA (Fig. 4A, lanes 5±8, and B). To con®rm that EKLF was
acting through its binding site at the proximal CACCC box in
the b-globin promoter (42), we introduced a C®G transversion at position ±87 to recreate a naturally occurring mutation
known to result in b-thalassemia (31). The HS2b clones with
the ±87 mutation HS2b(CACm) failed to respond to EKLF
and displayed a level of transcriptional activity similar to that
of the wild-type gene with no enhancer (Fig. 4A, lanes 9±12,
and B).
HS3 increased b-globin expression about 10-fold over the
level of the b-globin gene alone, in striking contrast to the
results seen with HS3 and the e-globin gene (Fig. 4A, compare
lanes 13±14 with lanes 1±4, and B). HS3 activates the b-globin
gene in an EKLF-independent manner (Fig. 4B) although
other workers have found that HS3 function requires EKLF
(see Discussion). Unlike HS2 and HS3, HS4 exerts a marginal
effect at most on b-globin gene expression, even in the
presence of EKLF (HS4b, Fig. 4A, lanes 15±18, and B).
Transcriptional activation by EKLF requires the EKLF
transactivation domain
A number of in vivo and in vitro approaches have demonstrated that the EKLF transactivation domain is required for
stimulation of the b-globin promoter through the recruitment
of chromatin remodeling complexes and probably other
accessory factors (8,37,41,43,44). To explore the role of
EKLF in HS2b transactivation, we transfected a HS2b cell
line with increasing amounts of expression plasmids for fulllength EKLF, the EKLF DBD alone, or an empty vector. Prior
work indicates that the stability of wild-type and deleted forms
of EKLF is similar (8,41). In contrast to the intact EKLF
polypeptide, the EKLF DBD fails to stimulate transcription
Nucleic Acids Research, 2003, Vol. 31, No. 4
Figure 5. EKLF stimulation of b-globin expression requires the EKLF
transactivation domain. An HS2b cell clone was transfected with pCI
EKLF, an expression plasmid for full-length EKLF, with pCI EKLFDPro,
which expresses the EKLF DBD, or pCI empty vector. (A) RNA was
isolated and b-globin RNA and EKLF RNA were measured by RNA protection. Actin RNA served as the loading control. Transfection mixtures contained 3, 5 or 9 mg of EKLF or EKLF DBD expression plasmid plus pCI to
a total of 10 mg. Empty, 10 mg of empty vector; control, human bone
marrow RNA. (B) The column graph depicts fold induction of b-globin
RNA normalized to actin RNA (black bars) and abundance of EKLF or
EKLF DBD RNA normalized to actin (gray bars). M, marker DNA in
nucleotides.
from HS2b even at high levels of expression (Fig. 5A and B).
These observations indicate that the EKLF transactivation
domain is required for transcriptional activation of the
b-globin gene on minichromosomes, and suggest that EKLF
also functions in this system by recruiting accessory factors to
the b-globin promoter (8,44).
The e- and b-globin genes confer different structures on
HS3
HS2 linked to the e-globin gene was highly accessible to
nucleases, and was required to remodel the chromatin
structure of the e-globin gene promoter to the transcriptionally
active state (24). Since active genes and regulatory elements
are typically found in open, nuclease-sensitive regions of
chromatin, we wished to investigate the structures of e- and
b-globin genes linked to the various HSs and the structures of
the HSs themselves. We also investigated whether the
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expression of EKLF in K562 cells could in¯uence the
structure of HS2 or HS3 because there is evidence implicating
ELKF in HS3 formation (27,28) and chromatin remodeling
(37,44).
Nuclei were isolated from cell lines carrying HS3e or HS4e
minichromosomes and, as controls, e-globin unlinked to a HS,
linked to HS2, or linked to a mutated form of HS2 (NFm)
which does not form a HS and fails to activate the e-globin
gene because the essential NF-E2 sites in HS2 have been
destroyed by clustered point mutations (24). The nuclei were
digested with AvaII or MscI and accessibility at the promoters
and the HSs was analyzed by Southern blotting and indirect
end labeling. Representative experiments are depicted in
Figure 6A and the results of multiple experiments presented
graphically in Figure 6C. Commensurate with the transcription results of Figure 3, restriction enzyme accessibility of the
e-globin promoter was not altered by HS3. Further, HS3 was
inaccessible when linked to the e-globin gene, with MscI
cutting of 2%, comparable to the HS2 NF-E2 mutant (9%),
and in contrast to over 60% accessibility for wild-type actively
transcribing eHS2. Expression of EKLF from a second
minichromosome did not affect this result (not shown).
Interestingly, HS4 and the linked e-globin gene promoter
were accessible to restriction enzyme cutting, even though
HS4 did not activate transcription from the gene (see Fig. 3B).
In parallel experiments, we tested accessibility of the
b-globin gene unlinked to a HS, or linked to HS2 or HS3 in
cell lines in which a second minichromosome carried either a
control or an EKLF-expressing cassette. Representative
experiments are depicted in Figure 6B and the results of
multiple experiments presented graphically in Figure 6C. HS2
and HS3 displayed similar levels of MscI accessibility of
~40% when linked to the b-globin gene and accessibility was
not affected by the expression of EKLF. HS4b does not
contain a suitable restriction site with which to measure
accessibility, but DNase I digestion revealed that HS4 does
form when linked to the b-globin gene (not shown). MscI also
cuts just 5¢ to the proximal CACCC box, and was used to
probe b-globin promoter structure. The b-globin promoter was
accessible to MscI whether or not it was linked to an HS and
whether or not it was transcriptionally active. Several
conclusions can be drawn from these results. First, a globin
promoter can be in a relatively open chromatin conformation
and not be transcriptionally active. Second, the accessibility
and structure of HS3 is strongly in¯uenced by the gene to
which it is linked. Third, although EKLF is required for
activation of b-globin transcription by HS2 in minichromosomes, in this assay it does not affect the structure of HS2 or of
the promoter. In addition, we ®nd no evidence that either the
structure or activity of HS3 is affected by EKLF expression in
K562 cells.
Structural and functional interactions between HS2 and
HS3
To begin addressing whether the LCR HSs interact with each
other in chromatin, we inserted both HS2 and HS3 upstream of
the b-globin gene (HS3+HS2b) in minichromosomes. To
control for promoter competition and potential spacing effects
on HS3 activity due to interposing HS2 between HS3 and the
b-globin gene, we also created HS3+HS2b minichromosomes
in which the HS2 tandem NF-E2 sites, or both CACCC sites,
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Nucleic Acids Research, 2003, Vol. 31, No. 4
Figure 6. The chromatin structure of HS3 depends on the linked globin gene. Nuclei of cells carrying various minichromosomes were prepared, digested with
the appropriate restriction enzyme cleaving within either the promoter or enhancer, and processed as described in Materials and Methods. (A) Representative
results obtained with e-globin clones with no enhancer, or linked to HS2, HS3 or HS4, or to HS2 with mutated NF-E2 sites (NFm). To analyze the promoters,
nuclei were incubated with AvaII and DNA cleaved secondarily with BglII. To analyze enhancers, nuclei were incubated with MscI and DNA cleaved
secondarily with EcoRV. MscI cleaves within both HS2 and HS3 (see Fig. 7A). Blots were hybridized to an XbaI±EcoRV probe. (B) Representative results
obtained with b-globin clones with no enhancer or linked to HS2 or HS3, and carrying a second EKLF-expressing minichromosome, or an empty vector. To
analyze the promoters, nuclei were incubated with MscI and cleaved secondarily with EcoRI. To analyze enhancers, nuclei were incubated with MscI and
cleaved secondarily with XcmI plus SphI. Blots were hybridized to a PmeI±SphI probe. (C) The mean percent accessibility of the promoters and enhancers
(determined three times on 2±5 clones) is presented graphically 6 SEM for the e-globin (black bars) and b-globin (gray bars) clones studied.
had been destroyed by clustered point mutations. These
mutations markedly impair HS2 function when linked to the
e-globin gene (24,35); they also strongly adversely affect HS2
function when positioned upstream of the b-globin gene (see
below). Figure 7A schematically depicts the HS3+HS2
constructs including the positions of transcription factor
binding sites of interest and the MscI restriction sites.
In the absence of EKLF, HS3+HS2b transcription differs
little from that of HS3b either in the presence or absence of
EKLF (Fig. 7B). However, HS3 and HS2 together resulted in a
synergistic stimulation of b-globin transcription in the
presence of EKLF. The sum of HS3b and HS2b RNA/actin/
copy number individually is 0.096 compared with 0.143 for
the HS3+HS2b minichromosome. These observations suggest
that HS3 and HS2 interact directly or indirectly to stimulate
transcription of the b-globin gene.
Before assaying the activity of constructs containing
mutated HS2 core elements joined to HS3, we ®rst con®rmed
that the clustered point mutations in the NF-E2 and CACCC
sites of HS2 impaired HS2 function when linked to the
b-globin gene as they do when linked to e-globin. Even in the
presence of EKLF, the NF-E2 mutation reduced the transcriptional activity of the HS2 b-globin construct by ~50%,
and the CACCC mutation essentially eliminated HS2 stimulation of b-globin transcription (Fig. 7B). Strikingly, when
either mutation is present in a HS3+HS2b construct, transcription of the b-globin promoter is reduced below the level
seen with HS3 alone, to ~30% of HS3b for the NF-E2 mutant
and to less than 15% for the CACCC mutant. These data
further support the ideas that HS2 and HS3 interact functionally in the context of the b-globin promoter and that a mutant
HS2 can dominantly interfere with the ability of HS3 to
activate the b-globin gene.
Since we observed structural interdependencies between the
HS2 and HS3 core elements and the b- and e-globin
promoters, we next asked whether we could also observe
structural interdependencies between juxtaposed HS2 and
HS3 core elements. Hence, we assayed MscI accessibility at
HS2 and HS3 linked to the b-globin gene, either alone or in
combination (Fig. 7C). MscI accessibility at HS3 in
HS3+HS2b is substantially altered compared with the accessibility of HS3 alone. The change in accessibility does not
require wild-type HS2 transactivation function (compare HS3
accessibility in the wild-type HS3+HS2 construct with its
accessibility in either the NF-E2 or the CACCC mutant
HS3+HS2 construct) and occurs even when the structure of
HS2 itself is altered by the NF-E2 mutation. These observations are consistent with the conclusion that HS2 and HS3
interact structurally.
DISCUSSION
The question of how the globin genes are regulated either
individually or collectively by the LCR HSs remains open. We
Nucleic Acids Research, 2003, Vol. 31, No. 4
1187
Figure 7. HS2 and HS3 structurally and functionally interact to stimulate b-globin transcription. (A) Depiction of MscI sites with respect to transcription
factor motifs within HS2 and HS3 (not drawn to scale). The 3 indicates which motifs in HS2 were mutated by clustered point mutations. (B) Graphical representation of the results of RNA protection assays performed on RNA from clones of HS2b, HS3b and HS3+HS2b as described in the legend to Figure 3. The
clones co-expressed EKLF from a second minichromosome (black bars) or had a vector-only minichromosome (gray bars). (C) Graphical representation of
analysis of the chromatin structure of HS2 and HS3 when alone or linked in minichromosomes. Restriction enzyme accessibility at the MscI sites present in
both HS2 (black bars) and HS3 (gray bars) was determined as described in the legend to Figure 6.
have begun experiments in replicating minichromosomes to
examine the interactions between the b-globin LCR HSs and
different globin promoters using a reassembly approach. Our
experiments indicate that the structure and function of HSs are
dependent on the other regulatory elements they interact with,
including the other HSs and globin gene promoters. We
propose that the basis of this variability is structural and that
the LCR HSs and globin promoters form a ¯uid transcriptional
complex whose overall conformation depends on the speci®c
components and transcription factors present. Recent, novel
in vivo experiments provide direct evidence of preferential and
close interaction between particular LCR HSs, in particular
HS2, HS3 and HS4, and the murine b-globin Hbb-b1 and
Hbb-b2 genes (45). Given that the sequence of the individual
HSs and organization of the LCR as a whole are highly
conserved within and across species in comparison with the
globin promoters (3), this model focuses attention on the
individual globin genes as key regulatory sites for stagespeci®c developmental regulation.
Individual LCR HSs, EKLF and the globin genes
Our experiments indicate preferential interdependencies in
structure and function between the e- and b-globin genes and
the HSs we tested in the K562 embryonic/fetal milieu. For
example, our previous studies of HS2 linked to the embryonic
e-globin gene on minichromosomes indicated that HS2 had a
highly accessible chromatin structure and transactivated the
e-globin gene over 100-fold (35). In contrast, and consistent
with experiments using stably integrated constructs in K562
cells (3), neither HS3 nor HS4 stimulated transcription from
the e-globin gene in episomes. We found that HS3 was highly
resistant to restriction enzyme cleavage when linked to the
e-globin gene, similar to its nuclease-resistant state in the
chromosomal locus in K562 cells (32,46). Others have
reported that HS3 function is dependent on EKLF (23,28).
The DNase I sensitivity of HS3 was also found to be
diminished following genetic ablation of EKLF in mice (27).
However, we found that EKLF overexpression alone was
insuf®cient to alter HS3 chromatin structure either on
minichromosomes or at the endogenous locus. The reason
that EKLF alone does not restore HS3 structure in K562 cells
may re¯ect differences in the transcription factor milieu
between K562 cells and hematopoietic cells. Possibly, an
ancillary factor(s) is missing in K562 cells relevant to
formation of HS3, and possibly to interaction between HS3
and the e-globin gene. Interestingly, HS4 does form both at the
chromosomal locus and on minichromosomes when linked to
the e-globin gene (not shown), but it does not activate
transcription of the gene in our experiments. This suggests
a component relevant to transcription activation by HS4
is de®cient or lacking in K562 cells. Work by others
using transgenic mice suggests that HS4 function may require
the regulatory environment of de®nitive erythroid cells
(10,12).
HS2, HS3 and HS4 all formed nuclease-accessible structures when linked to the b-globin gene. Transactivation of the
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Nucleic Acids Research, 2003, Vol. 31, No. 4
b-globin gene by HS2 was EKLF dependent. Both the
b-globin promoter CACCC box and the EKLF activation
domain are required for this activation. However, transactivation did not re¯ect an alteration of HS2 chromatin structure
mediated by EKLF. Therefore, EKLF may stabilize
promoter±HS2 interactions or may participate in recruiting
basal transcription factors required for b-globin activation
(44). HS3 does not appear to be a target for EKLF in K562
cells (regardless of whether it is linked to the e- or b-globin
gene), consistent with previous work (30). We found that the
strong transactivation of the b-globin gene by HS3 was EKLF
independent, while, similarly to HS2, its chromatin structure
was unaltered by EKLF. Interestingly, HS3 was relatively
sensitive to restriction enzyme cleavage when linked to the
b-globin gene in contrast to its structure in the endogenous
K562 chromosome and when linked to the e-globin gene.
Perhaps the closer linkage of the b-globin gene and HSs on
minichromosomes (2 kb in contrast to 60 kb) in¯uences the
structure of HS3. Once HS3 structure is formed, it may no
longer need EKLF to interact with the b-globin gene.
The differential interactions we observed between the LCR
HSs and the b- and e-globin genes in K562 cells contrast with
HS deletion studies in the mouse chromosomal globin locus
where preferential interactions between particular HSs and
globin promoters are not obvious (reviewed in 47). However,
we suggest that preferential but non-exclusive interactions
between the HSs and globin promoters and among the HSs
may become apparent when the HSs are tested individually
rather than in the context of the globin locus. In the intact
LCR, preferential interactions could be masked by complementation among the linked HSs. The differences between
promoter±HS interactions may be most easily understood in
terms of an interaction model in which the particular arrays of
transcription activators within the HSs and promoters create
complementary structures that mediate preferential crosstalk
between these elements (24,48). Possible candidate molecules
for effecting such communication include the erythroid factors
GATA-1, EKLF and NF-E2 (7,8,49).
Interactions among the LCR HSs
Since other laboratories have reported synergistic transcriptional interactions between HSs and the b- and g-globin
promoters (50±54), we linked HS3 and HS2 to the b-globin
gene in minichromosomes. We observed modest synergistic
stimulation of b-globin transcription by HS3+HS2 in the
presence of forced expression of EKLF. Moreover, when we
replaced the wild-type HS2 in the HS3+HS2b minichromosome with either of two different HS2 elements containing
clustered point mutations that impair the ability of HS2 to
transactivate the b- and e-globin promoters, the level of
transcription from the HS3+HS2b-globin promoter fell below
the level seen with HS3 alone even though HS3 by itself
stimulates transcription more strongly than HS2. The
dominant interference of HS2 in activation by HS3 supports
the idea that HS3 and HS2 interact with one another
functionally. This interaction apparently does not require
NF-E2 or the CACCC sites in HS2.
We also observed, somewhat surprisingly, that linking HS3
to either a wild-type or a mutated HS2 element decreased the
accessibility of HS3 to MscI digestion in isolated nuclei. We
infer from this reproducible alteration in accessibility that a
structural change in HS3 has occurred, although from this data
we cannot determine the nature of the alteration: for example,
the association of additional factors with the complex might
occlude the site, or structural interaction with HS2 might
reorganize HS3 moving the MscI site into the interior of the
complex. We cannot exclude the possibility that the reduction
of HS3 accessibility simply results from the steric effect of
juxtaposing HS2 to HS3 rather than from alterations in the
structure of HS3; however, Lee et al. (30) observed that HS2
facilitated the recruitment of EKLF to a linked HS3, which
also suggests that structural communication between the two
elements occurs. It will be important to determine whether
such interactions also occur with physiological spacing
between the cores, inclusion of sequences ¯anking them,
and/or a chromosomal context.
Promoter±HS communication
In this work we provide further evidence of communication
between HSs and globin promoters since the extent of
restriction enzyme accessibility of HS3 varies markedly
depending on whether it is linked to the e- or b-globin gene.
These data extend our previous observation that mutations in
the promoter of the e-globin gene alter the structure of a
linked HS2 element (24). Thus, there also appear to be
structural interdependencies between HSs and different
globin promoters.
Although the different HSs clearly have some ability to
complement each other functionally in the context of the intact
LCR (17±20), they also appear to display preferential
interactions with the different b-like globin genes that may
be important for correct developmental regulation. Our studies
were conducted in the fetal/embryonic milieu of K562 cells,
but at other stages of development the interactions we
observed are likely to be modulated. A comparison of our
results with those obtained in an environment where the
b-globin gene is actively transcribed, such as murine MEL
cells, would be desirable. However, EBV episomes become
chromosomally integrated in rodent cells, requiring an entirely
new approach, perhaps using BPV episomes, to such
experiments. In our studies, the ability of HS2 to alter the
structure of a linked HS3 and of a defective HS2 to impair
the transactivating potential of a linked HS3 indicate that
HS2, HS3 and the b-globin promoter interact with one
another structurally and functionally, perhaps forming a
transcription enhancing complex including HSs and a
target promoter which would then recruit chromatin remodeling and modifying complexes, perhaps as part of the
transcription apparatus, to the promoter. Conservation of the
structure of the LCR would then be expected to re¯ect
important structural interactions not only among the HSs
themselves, but also between the HSs and the globin
promoters.
ACKNOWLEDGEMENTS
We thank Dr James Bieker for kindly providing EKLF
expression vectors and antibodies, and Drs Jane Little, Cecelia
Trainor and James Bieker for critical reading of the
manuscript.
Nucleic Acids Research, 2003, Vol. 31, No. 4
REFERENCES
1. Stamatoyannopoulos,G. and Grosveld,F. (2001) Hemoglobin switching.
In Stamatoyannopoulos,G., Majerus,P.W., Perlmutter,R.M. and
Varmus,H. (eds), The Molecular Basis of Blood Diseases. W.B.
Saunders, Philadelphia, PA, pp. 135±182.
2. Reik,A., Telling,A., Zitnik,G., Cimbora,D., Epner,E. and Groudine,M.
(1998) The locus control region is necessary for gene expression in the
human b-globin locus but not the maintenance of an open chromatin
structure in erythroid cells. Mol. Cell. Biol., 18, 5992±6000.
3. Hardison,R., Slightom,J.L., Gumucio,D.L., Goodman,M., Stojanovic,N.
and Miller,W. (1997) Locus control regions of mammalian b-globin gene
clusters: combining phylogenetic analyses and experimental results to
gain functional insights. Gene, 205, 73±94.
4. Forrester,W.C., Thompson,C., Elder,J.T. and Groudine,M. (1986) A
developmentally stable chromatin structure in the human b-globin gene
cluster. Proc. Natl Acad. Sci. USA, 83, 1359±1363.
5. Tuan,D., Solomon,W., Li,Q. and London,I.M. (1985) The "beta-likeglobin" gene domain in human erythroid cells. Proc. Natl Acad. Sci.
USA, 82, 6384±6388.
6. Jackson,P.D., Evans,T., Nickol,J.M. and Felsenfeld,G. (1989)
Developmental modulation of protein binding to b-globin gene
regulatory sites within chicken erythrocyte nuclei. Genes Dev., 3,
1860±1873.
7. Andrews,N.C., Erdjument-Bromage,H., Davidson,M.B., Tempst,P. and
Orkin,S.H. (1993) Erythroid transcription factor NF-E2 is a
haematopoietic-speci®c basic-leucine zipper protein. Nature, 362,
722±728.
8. Bieker,J.J. and Southwood,C.M. (1995) The erythroid Kruppel-like
factor transactivation domain is a critical component for cell-speci®c
inducibility of a b-globin promoter. Mol. Cell. Biol., 15, 852±860.
9. Levings,P.P. and Bungert,J. (2002) The human beta-globin locus control
region. Eur. J. Biochem., 269, 1589±1599.
10. Fraser,P., Pruzina,S., Antoniou,M. and Grosveld,F. (1993) Each
hypersensitive site of the human b-globin locus control region confers a
different developmental pattern of expression on the globin genes.
Genes Dev., 7, 106±113.
11. Navas,P.A., Peterson,K.R., Li,Q., Skarpidi,E., Rohde,A., Shaw,S.E.,
Clegg,C.H., Asano,H. and Stamatoyannopoulos,G. (1998)
Developmental speci®city of the interaction between the locus control
region and embryonic or fetal globin genes in transgenic mice with an
HS3 core deletion. Mol. Cell. Biol., 18, 4188±4196.
12. Navas,P.A., Peterson,K.R., Li,Q., McArthur,M. and
Stamatoyannopoulos,G. (2001) The 5¢HS4 core element of the human
b-globin locus control region is required for high-level globin gene
expression in de®nitive but not in primitive erythropoiesis. J. Mol. Biol.,
312, 17±26.
13. Bungert,J., Dave,U., Lim,K.C., Lieuw,K.H., Shavit,J.A., Liu,Q. and
Engel,J.D. (1995) Synergistic regulation of human b-globin gene
switching by locus control region elements HS3 and HS4. Genes Dev., 9,
3083±3096.
14. Bungert,J., Tanimoto,K., Patel,S., Liu,Q., Fear,M. and Engel,J.D. (1999)
Hypersensitive site 2 speci®es a unique function within the human
b-globin locus control region to stimulate globin gene transcription.
Mol. Cell. Biol., 19, 3062±3072.
15. Li,G., Lim,K.C., Engel,J.D. and Bungert,J. (1998) Individual LCR
hypersensitive sites cooperate to generate an open chromatin domain
spanning the human beta-globin locus. Genes Cells, 3, 415±429.
16. Ellis,J., Tan-Un,K.C., Harper,A., Michalovich,D., Yannoutsos,N.,
Philipsen,S. and Grosveld,F. (1996) A dominant chromatin-opening
activity in 5¢ hypersensitive site 3 of the human b-globin locus control
region. EMBO J., 15, 562±568.
17. Fiering,S., Epner,E., Robinson,K., Zhuang,Y., Telling,A., Hu,M.,
Martin,D.I.K., Enver,T., Ley,T.J. and Groudine,M. (1995) Targeted
deletion of 5¢HS2 of the murine b-globin LCR reveals that it is not
essential for proper regulation of the b-globin locus. Genes Dev., 9,
2203±2213.
18. Hug,B.A., Wesselschmidt,R.L., Fiering,S., Bender,M.A., Epner,E.,
Groudine,M. and Ley,T.J. (1996) Analysis of mice containing a targeted
deletion of b-globin locus control region 5¢ hypersensitive site 3.
Mol. Cell. Biol., 16, 2906±2912.
19. Peterson,K.R., Clegg,C.H., Navas,P.A., Norton,E.J., Kimbrough,T.G.
and Stamatoyannopoulos,G. (1996) Effect of deletion of 5¢HS3 or 5¢HS2
of the human b-globin locus control region on the developmental
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
1189
regulation of globin gene expression in b-globin locus yeast arti®cial
chromosome transgenic mice. Proc. Natl Acad. Sci. USA, 93, 6605±6609.
Bender,M.A., Roach,J.N., Halow,J., Close,J., Alami,R., Bouhassira,E.E.,
Groudine,M. and Fiering,S.N. (2001) Targeted deletion of 5¢HS1 and
5¢HS4 of the b-globin locus control region reveals additive activity of the
DNaseI hypersensitive sites. Blood, 98, 2022±2027.
Bender,M.A., Mehaffey,M.G., Telling,A., Hug,B., Ley,T.J., Groudine,M.
and Fiering,S. (2000) Independent formation of DnaseI hypersensitive
sites in the murine b-globin locus control region. Blood, 95, 3600±3604.
Reitman,M., Lee,E., Westfall,H. and Felsenfeld,G. (1993) An enhancer/
locus control region is not suf®cient to open chromatin. Mol. Cell. Biol.,
13, 3990±3998.
Tewari,R., Gillemans,N., Harper,A., Wijgerde,M., Zafarana,G.,
Drabek,D., Grosveld,F. and Philipsen,S. (1996) The human b-globin
locus control region confers an early embryonic erythroid-speci®c
expression pattern to a basic promoter driving the bacterial lacZ gene.
Development, 122, 3991±3999.
McDowell,J.C. and Dean,A. (1999) Structural and functional cross-talk
between a distant enhancer and the e-globin gene promoter shows
interdependence of the two elements in chromatin. Mol. Cell. Biol., 19,
7600±7609.
Nuez,B., Michalovich,D., Bygrave,A., Ploemacher,R. and Grosveld,F.
(1995) Defective haematopoiesis in fetal liver resulting from inactivation
of the EKLF gene. Nature, 375, 316±318.
Perkins,A.C., Sharpe,A.H. and Orkin,S.H. (1995) Lethal b-thalassaemia
in mice lacking the erythroid CACCC- transcription factor EKLF.
Nature, 375, 318±322.
Wijgerde,M., Gribnau,J., Trimborn,T., Nuez,B., Philipsen,S., Grosveld,F.
and Fraser,P. (1996) The role of EKLF in human beta-globin gene
competition. Genes Dev., 10, 2894±2902.
Gillemans,N., Tewari,R., Lindeboom,F., Rottier,R., de Wit,T.,
Wijgerde,M., Grosveld,F. and Philipsen,S. (1998) Altered DNA-binding
speci®city mutants of EKLF and Sp1 show that EKLF is an activator of
the b-globin locus control region in vivo. Genes Dev., 12, 2863±2873.
Tewari,R., Gillemans,N., Wijgerde,M., Nuez,B., von Lindern,M.,
Grosveld,F. and Philipsen,S. (1998) Erythroid Kruppel-like factor
(EKLF) is active in primitive and de®nitive erythroid cells and is
required for the function of 5¢HS3 of the b-globin locus control region.
EMBO J., 17, 2334±2341.
Lee,J.S., Lee,C.H. and Chung,J.H. (1999) The b-globin promoter is
important for recruitment of erythroid Kruppel-like factor to the locus
control region in erythroid cells. Proc. Natl Acad. Sci. USA, 96,
10051±10055.
Donze,D., Townes,T.M. and Bieker,J.J. (1995) Role of erythroid
Kruppel-like factor in human g- to b-globin gene switching. J. Biol.
Chem., 270, 1955±1959.
Dhar,V., Nandi,A., Schildkraut,C.L. and Skoultchi,A.I. (1990) Erythroidspeci®c nuclease-hypersensitive sites ¯anking the human b-globin
domain. Mol. Cell. Biol., 10, 4324±4333.
Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning:
A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY.
Yates,J.L., Warren,N. and Sugden,B. (1985) Stable replication of
plasmids derived from Epstein-Barr virus in various mammalian cells.
Nature, 313, 812±815.
Gong,Q.H., McDowell,J.C. and Dean,A. (1996) Essential role of NF-E2
in remodeling of chromatin structure and transcriptional activation of the
e-globin gene in vivo by 5¢ hypersensitive site 2 of the b-globin locus
control region. Mol. Cell. Biol., 16, 6055±6064.
Miller,I.J. and Bieker,J.J. (1993) A novel, erythroid cell-speci®c murine
transcription factor that binds to the CACCC element and is related to the
Kruppel family of nuclear proteins. Mol. Cell. Biol., 13, 2776±2786.
Brown,R.C., Pattison,S., van Ree,J., Coghill,E., Perkins,A., Jane,S.M.
and Cunningham,J.M. (2002) Distinct domains of erythroid Kruppel-like
factor modulate chromatin remodeling and transactivation at the
endogenous b-globin gene promoter. Mol. Cell. Biol., 22, 161±170.
Gong,Q. and Dean,A. (1995) Enhancer dependent transcription of the
human e-globin gene on a stably maintained minichromosome. In
Stamatoyannopoulos,G. (ed.), Molecular Biology of Hemoglobin
Switching. Intercept, Andover, UK, pp. 279±288.
Dignam,J.D., Lebowitz,R.M. and Roeder,R.G. (1983) Accurate
transcription initiation by RNA polymerase II in a soluble extract from
isolated mammalian nuclei. Nucleic Acids Res., 11, 1475±1489.
1190
Nucleic Acids Research, 2003, Vol. 31, No. 4
40. Ouyang,L., Chen,X. and Bieker,J.J. (1998) Regulation of erythroid
Kruppel-like factor (EKLF) transcriptional activity by phosphorylation of
a protein kinase casein kinase II site within its interaction domain. J. Biol.
Chem., 273, 23019±23025.
41. Chen,X. and Bieker,J.J. (1996) Erythroid Kruppel-like factor (EKLF)
contains a multifunctional transcriptional activation domain important
for inter- and intramolecular interactions. EMBO J., 15, 5888±5896.
42. Perkins,A. (1999) Erythroid Kruppel like factor: from ®shing expedition
to gourmet meal. Int. J. Biochem. Cell Biol., 31, 1175±1192.
43. Zhang,W. and Bieker,J.J. (1998) Acetylation and modulation of erythroid
Kruppel-like factor (EKLF) activity by interaction with histone
acetyltransferases. Proc. Natl Acad. Sci. USA, 95, 9855±9860.
44. Armstrong,J.A., Bieker,J.J. and Emerson,B.M. (1998) A SWI/SNFrelated chromatin remodeling complex, E-RC1, is required for
tissue-speci®c transcriptional regulation by EKLF in vitro. Cell, 95,
93±104.
45. Carter,D., Chakalova,L., Osborne,C.S., Dai,Y. and Fraser (2002)
Long-range chromatin regulatory interactions in vivo. Nature Genet., 32,
623±626.
46. Guy,L.G., Mei,Q., Perkins,A.C., Orkin,S.H. and Wall,L. (1998)
Erythroid Kruppel-like factor is essential for b-globin gene expression
even in absence of gene competition, but is not suf®cient to induce
the switch from g-globin to b-globin gene expression. Blood, 91,
2259±2263.
47. Bulger,M. and Groudine,M. (1999) Looping versus linking: toward a
model for long-distance gene activation. Genes Dev., 13, 2465±2477.
48. Huber,M.C., Jagle,U., Kruger,G. and Bonifer,C. (1997) The
developmental activation of the chicken lysozyme locus in transgenic
mice requires the interaction of a subset of enhancer elements with the
promoter. Nucleic Acids Res., 25, 2992±3000.
49. Evans,T. and Felsenfeld,G. (1989) The erythroid-speci®c transcription
factor Eryf1: a new ®nger protein. Cell, 58, 877±885.
50. Jackson,J.D., Miller,W. and Hardison,R.C. (1996) Sequences within and
¯anking hypersensitive sites 3 and 2 of the b-globin locus control region
required for synergistic versus additive interaction with the e-globin gene
promoter. Nucleic Acids Res., 24, 4327±4335.
51. Jackson,J.D., Petrykowska,H., Philipsen,S., Miller,W. and Hardison,R.
(1996) Role of DNA sequences outside the cores of DNase
hypersensitive sites (HSs) in functions of the b-globin locus control
region. Domain opening and synergism between HS2 and HS3. J. Biol.
Chem., 271, 11871±11878.
52. Bresnick,E.H. and Tze,L. (1997) Synergism between hypersensitive sites
confers long-range gene activation by the b-globin locus control region.
Proc. Natl Acad. Sci. USA, 94, 4566±4571.
53. Sargent,T.G., DuBois,C.C., Buller,A.M. and Lloyd,J.A. (1999) The roles
of 5¢-HS2, 5¢-HS3, and the g-globin TATA, CACCC, and stage selector
elements in suppression of b-globin expression in early development.
J. Biol. Chem., 274, 11229±11236.
54. Molete,J.M., Petrykowska,H., Bouhassira,E.E., Feng,Y.Q., Miller,W. and
Hardison,R.C. (2001) Sequences ¯anking hypersensitive sites of the
b-globin locus control region are required for synergistic enhancement.
Mol. Cell. Biol., 21, 2969±2980.