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INTRODUCTION |
The human
-globin gene domain at chromosomal position 11p15.5
consists of a cluster of globin genes that are expressed only in
erythroid cells. The genes are arranged
5'-HBE-HBG2-HBG1-HBD-HBB-3' from the centromere to the telomere. The genes are temporally expressed
in the order of their array along the chromosome, with
-globin
(encoded by HBE) produced embryonically, G
-
and A
-globin (encoded by HBG2 and
HBG1) produced fetally, and
- and
-globin (encoded by
HBD and HBB) produced in the adult. Controlled expression of the genes occurs through proximal regulators, including the promoters of the individual genes, and distal elements, such as the
dominant regulatory region known as the locus control region (LCR)1 (1-4). The LCR spans
at least 17 kilobases of DNA, located 23 to 6 kilobases upstream of the
-globin gene, and contains 5 DNase I-hypersensitive sites (HS1-5)
(5-7). The LCR is defined by its ability to confer high level,
tissue-specific expression of linked genes at all sites of integration
examined in transgenic mice (8). It is a powerful enhancer (8-11).
Analysis of LCR deletions in thalassemic patients (12, 13) and
experiments testing LCR function at ectopic sites (e.g. 14, 15) have been interpreted as showing a domain-opening activity, but
this has not been seen in directed deletions of the LCR (10, 11). Thus,
all the functions of the LCR have not been conclusively defined (16),
but it is clear that it is a major cis-regulatory element for the
-like globin gene cluster. DNA fragments containing individual HSs
can produce some of the effects of the LCR (17-20), and growing
evidence supports the model that multiple HSs work together in a
holocomplex at the LCR (9, 15, 21-25). A full set of cis-regulatory
sites and the protein(s) that works at them is not yet known either for
the function of an individual HS or for its interaction with other sites.
One of the most potent components of the LCR is HS2, which can produce
high level, position-independent expression of linked genes in
transgenic mice (17, 20, 26) and stably transfected erythroid cell
lines, including both human K562 cells and mouse erythroleukemia (MEL)
cells (21, 26, 27). It can also act without stable integration into
chromosomes, conferring both strong enhancement (28, 29) and
heme-inducibility (30-32) on linked globin genes in transiently
transfected K562 cells. The core of HS2 is defined as the smallest
region conferring position-independent expression of the
-globin
gene; it is contained within a 374-base pair
HindIII-XbaI fragment (26, 33). Simultaneous
alignment of multiple DNA sequences of the
-globin domain from
mammals shows several conserved blocks, many of which have been
confirmed as protein binding sites needed for the function of HS2 (4, 34). A particularly dense cluster of sites, spaced at 10-base pair
intervals within a 100-base pair segment of the HS2 core, is suggestive
of a contiguous array of proteins on the same face of the DNA helix. A
dimer of MAREs, or Maf-responsive
elements (35), is crucial for the enhancement and
heme-inducibility by HS2 in K562 cells (30, 31) as well as high level
expression in transgenic mice (26, 33, 36). The MAREs are binding sites for AP1, NFE2 (37-39), Nrf1 (40)/LCRF1 (41)/TCF11 (42), Nrf2 (43), and Bach1 (44). Most of these proteins function as heterodimers with small Maf proteins; hence, the binding site has been named for
this common component. The MAREs are not sufficient for full-level enhancement (26, 27, 31, 32), indicating that other proteins are also
functioning at HS2. In particular, GATA1 and/or GATA2 (26, 33, 45, 46),
basic helix-loop-helix proteins and other E box-binding proteins (32),
HS2NFE5 (47), and proteins binding to the highly conserved CAC motif
(33) have all been implicated by a combination of mutagenesis, in
vivo footprinting, in vitro binding coupled with
antibody studies, and sequence conservation. Additional conserved sites
are found throughout and beyond the HS2 core (4), and many of these
regions have also been implicated in function of the LCR (33, 48,
49).
To better understand the contribution of the several conserved
sequences in the HS2 core to enhancement in erythroid cells, we
developed methods using cationic lipids to transiently transfect several erythroid cell lines (50). This has allowed us to test constructs containing promoters from several
-like globin genes in
murine MEL cells (which produce
-globin (51)), human K562 cells
(which produce
- and
-globin (52, 53)), and human erythroleukemia
(HEL) cells (which produce mainly
-globin (54)). The results
identify cis-acting sequences within the HS2 core that negatively
regulate expression of linked
-like globin genes in erythroid cell
lines and in normal human adult erythroid cells. The negative effect is
seen both in transiently transfected cells and after integration into
stably transformed cells.
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EXPERIMENTAL PROCEDURES |
Transient Transfection of Erythroid Cells--
The cationic
lipid reagent Tfx50 (from Promega) was used to transiently transfect
MEL, K562, and HEL cells (50) following the manufacturer's protocol
for suspension cells. Optimal transfection was obtained at a 2:1 ratio
(charge to mass) of cationic lipid to DNA. The reagent and DNA remained
in the cell culture for 48 h, at which point the cells were
harvested. Three different experimental designs were used to examine
the level of expression in transient transfections. In the
first, each plasmid containing a reporter gene was assayed as a
titration of DNA mass from 0.25 µg up to a maximum of 8.0 µg
(maintaining the 2:1 charge to mass ratio), with the results reported
as luciferase activity in relative light units (RLU)/s. In the
second, plasmids were transfected in triplicate at the single DNA mass
most frequently seen to be optimal for enhancement in each cell line,
which was 2.0 µg for MEL cells and 3.0 µg for K562 and HEL cells.
The resulting luciferase activities were normalized to the amount of
total protein in each sample, which was determined by the Bio-Rad BCA
microprotein assay. In this design, Student's t test was
used to determine the probability that paired samples did not belong to
distinct, nonoverlapping data sets. Our initial attempts to use
pRSVlacZ as a cotransfection control were unsuccessful, since the
amount of pRSVlacZ required to obtain activity above the background
were incompatible with experimentally determined transfection
conditions, which limited test DNA to a maximum of 2 µg total in MEL
cells (50).
Subsequent testing showed that small amounts of a cotransfection
control vector expressing the luciferase gene from Renilla (sea pansy, Promega) provided measurable activity without interfering unacceptably with the test plasmid. Thus the third design was to
include the Renilla luciferase cotransfection control
plasmid in the following amounts: for MEL cells, 1.75 µg of test
plasmid plus 0.25 µg of control plasmid, and for K562 and HEL cells,
1.9 µg of test plasmid and 0.1 µg of control plasmid.
Normal human adult erythroid cells (hAEC) were cultured from human
peripheral blood using the two-phase system of Fibach et al.
(55). They were transiently transfected by electroporation using the
methods described in Li et al. (56).
Stable Transfection of MEL Cells--
MEL cells (1 × 106) were stably transfected using the Tfx50 reagent to
introduce 1.5 µg of linearized test plasmid plus 0.5 µg of pSV2neo,
a neomycin phosphotransferase expression vector. One day after
transfection, G418 was added to the culture at 0.6 mg/ml. Pools of
stably transfected cells were harvested after 2 weeks of growth under
selection to measure luciferase activity. Pools of transfected cells
were induced by growth in 4 mM hexamethylene bisacetamide
(HMBA) for 5 days; uninduced cultures were maintained for the same
period in the absence of inducer.
Luciferase Assay--
As described previously (32), up to 20 µl of cellular extract was assayed in 100 µl of Promega luciferase
assay reagent.
Plasmid Construction--
Luciferase constructs containing the
rabbit
-globin promoter from
573 to +85, without (
luc or
pBS
-luciferase.4) or with the human HS2 core (
HS2 or
pBS
-luciferase-hHS2HX) have been described previously (48, 57). The
human HS2 core is the HindIII-XbaI fragment,
HUMHBB positions 8486-8860. The
-globin-luciferase reporter
constructs contained the mouse
-major globin gene
(Hbb-b1) promoter sequence from
106 to + 26 (
106),
340 to +26 (
340) or the HBB promoter from
374 to +45
(h
) fused to the luciferase-coding region of pGL2Basic (Promega)
(58). The human HS2 core was added by subcloning it initially into
pBluescriptII KS
(Stratagene), excising it by digestion with
KpnI and SacI and ligating it to these same
restriction cleavage sites within the
-globin-luciferase plasmids to
generate
106HS2 and
340HS2. Plasmid
luc contains the human
A
-globin gene promoter from,
260 to +35, fused to the
luciferase coding region of pGL3Basic (Promega), and plasmid
HS2 has
in addition the KpnI to BglII DNA fragment
(HUMHBB positions 7764-9218) containing HS2 (47).
HX and BX contain
the HindIII-XbaI and BalI-XbaI fragments of HS2, respectively.
Constructs containing the human MAREs or multimers of the HS2 E box
were made by inserting oligonucleotides, listed below, that had
flanking sequence to allow directional cloning (via HindIII
and SpeI sites) into either the
- or
-luciferase vectors.
Deletion Series through HS2--
Beginning with the
HindIII-XbaI fragment containing the human HS2
core cloned into pBluescript II KS
(pBSHS2HX), the desired deletion
fragments were obtained by restriction digestion or PCR amplification
and cloned into pBS
-luciferase.4 (48). The 5' deletion series
contained four constructs: BX (BalI to XbaI), MX
(Mares to XbaI), 8701X, and 8762X; the 5' ends are at HUMHBB positions 8568, 8658, 8701, and 8762 (respectively), and all 3' ends
are at 8860. The 3' deletion series contained four constructs: H8750
(HindIII-8750), HCAC (HindIII-CAC), H8650
(HindIII-8650), and HB (HindIII-BalI);
the 3' ends are at HUMHBB positions 8750, 8687, 8651, and 8568 (respectively), and all 5' ends are at 8486. The BX fragment of HS2 was
directionally cloned into the
luc plasmid by excising it from
pBSHS2HX with MscI and PstI and ligating it into
luc at SmaI and PstI sites to generate
luc-Bal-Xba. The BX fragment was also inserted into the
106
luciferase expression plasmid by excising it from
luc-Bal-Xba with
PstI and SpeI, cloning it into pBluescriptIIKS
at the same restriction sites, excising again with SacI and
BssHII, and ligating it into SacI and
MluI sites in the
106 plasmid. The other fragments were
amplified by PCR using the following primers (sequences are listed
below): MX by primers "human MAREs" and T7; 8701X by primers
8701/BamHI and T7; 8762X by primers 8762 and T7; H8750 by
primers 8750/PstI and T3; HCAC by primers
CAC/PstI and T3; H8650 by primers 8650/PstI and
T3; HBalI by primers BalI and T3. The resulting
PCR products were digested with appropriate restriction enzymes to
allow directional cloning into pBS
-luciferase.4. Three deletions
between the HindIII and BalI restriction sites
were generated that corresponded at their 5' ends to conserved
sequences within the HS2 core. The template for amplification was the
pBSHS2HX vector. All three constructs in this deletion series had the
same sequences at their 3' end, generated by using the T3 primer of
pBluescript. PCR products were prepared for subcloning into the
106-luciferase vector by digestion with restriction endonucleases.
KpnI was introduced in the sequence of the forward primer,
and SacI is found in the polylinker between the 3' end of
the HS2 core and the T3 primer.
Oligonucleotides--
The sequence of the top strand of the
duplex oligonucleotides used in cloning or the single-stranded
oligonucleotide used for PCR are as follows. 8701 E box multimer,
agctAGGCTGAGAACATCTGGGCACTAAGGCTGAGAACATCTGGGCACTAAGGCTGAGAACATCTGGGCACTAAGGCTGAGAACATCTGGGCACTAAGGCTGAGAACATCTGGGCA; human MAREs, ctagATGCTGAGTCATGATGAGTCATG;
rabbit MAREs,
ctagCAGTGCTGAGTCATGCTGAGTCATGTTGAGTCATGCTG; 8701/BamHI, caggatccGTGCCCAGATGTTCTCAG; 8762, ctagAGGGCAGATGGCAA; 8750/PstI,
aactgcagCCTGTAAGCATCCTGCTG; CAC/PstI
aactgcagGGCACACACCCTAAGC; 8650 gatcTTGCTGTGCTTGAGC; BalI,
agctCTGGCCAGAACTGCTC; CR2,
atggtaccTTAGTTCCTGTTACATTT; CR3,
atggtaccGTGTCTCCATTAGTGACC; CR4,
atggtaccTCCCATAGTCCAAG.
Nucleotides added at the 5' ends of the sequence are shown in
lowercase. Consensus sequences for binding sites, conserved regions, or
restriction cleavage sites are underlined.
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RESULTS |
Weak Enhancement by HS2 in MEL Cells--
Using cationic lipids to
deliver DNA to erythroid cells lines for transient expression, we were
surprised to find that the effects of HS2 on enhancement were quite
modest in MEL cells. A series of luciferase reporter gene plasmids
(Fig. 1A), driven by promoters
from either the rabbit embryonic HBE gene or the mouse
fetal/adult Hbb-b1 gene (encoding the
-major globin),
were transfected into MEL and K562 cells at increasing concentrations of DNA to find optimal conditions for enhancement. Comparison of the
resulting luciferase activities between plasmids with and without the
HS2 core shows the expected robust enhancement by HS2 for both the
HBE and the Hbb-b1 promoters in K562 cells (50- and 30-fold in this experiment, Fig. 1, B and C).
However, enhancement in MEL cells is substantially lower, being only
12- and 3-fold for the HBE and Hbb-b1 promoters,
respectively (Fig. 1, B and C). Although the
measured fold enhancement varies in different experiments, comparisons
of transfections done at the same time invariably show a lower level of
enhancement in MEL than in K562 cells. Also, as will be shown later,
this modest effect of the HS2 core in MEL cells is seen in several
different transfection protocols. Furthermore, a similar response from
the promoters of rabbit, mouse, or human globin genes to enhancers
containing fragments of the human HS2 core suggests that
species-specific effects are not evident.

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Fig. 1.
Comparison of HS2 enhancement in transiently
transfected K562 and MEL cells. A, diagrams of plasmids
illustrate the different promoters used (from the rabbit HBE
gene encoding -globin and the mouse Hbb-b1 gene encoding
the -major globin) as well as the different poly(A) signals from
HBE ( ) or SV40 (SV). The luciferase cDNA in these
reporter gene plasmids is from pGL2Basic (Promega). B-D,
panels comparing HS2 enhancement in K562 (left)
and MEL cells (right) on the HBE promoter
(B) or the Hbb-b1 promoter from 340
(C), or 106 (D) to +26. An increasing mass of
DNA was transfected into the cells, and the resulting luciferase
activity is plotted. Fold enhancement over the enhancerless construct
level of expression is shown in each box.
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The data in Fig. 1 show that the transfected plasmid templates were
active, even in cells in which the endogenous homologous genes are
inactive. In particular, the plasmid with an HBE promoter without an enhancer is as active in MEL cells, which do not produce
y-globin from the endogenous Hbb-y locus, as
in K562 cells, which produce
-globin from the endogenous
HBE gene (800 RLU/s in K562 and 500 RLU/s in MEL). However,
the response to the HS2 core was considerably lower in transiently
transfected MEL cells. Likewise, the plasmid with an Hbb-b1
promoter responded better to the HS2 core enhancer in K562 cells, where
the endogenous homolog HBB is inactive, than in MEL cells.
The Hbb-b1 promoter used for the data in Fig. 1C
extended to nucleotide position
340 (with respect to the cap), and
thus contained some sequences implicated in negative regulation of this
gene (59, 60). Deletion of these negative elements did not restore
enhancement in MEL cells, since the Hbb-b1 promoter
extending only to
106 still showed a 6-fold reduction in enhancement
in MEL cells compared with K562 cells (Fig. 1D). Thus, these
results indicate that the reduced enhancement in MEL cells is not
related to the type of globin gene promoter used or the activity of the
endogenous homologous genes.
The limited enhancement by the HS2 core in MEL cells is not an artifact
of using only parts of the genes. The plasmid
-luc (Fig.
1A) was designed to include almost all of the HBE
gene (57, 61). It has the rabbit HBE promoter segment
extending from
573 to +85, which includes multiple positive and
negative elements (62) that are homologous to those of the human
HBE gene (56, 63, 64). Since the luciferase-coding region
replaces a segment extending from the 3' end of exon 1 to almost the 3'
end of exon 2, the reporter gene retains most of the HBE
exons, intron 2, and an extensive 3'-flanking region. The difference in
enhancement between the two cell lines is seen both for expression
plasmids containing the promoter and internal and 3'-flanking regions
(of HBE) as well as for plasmids containing only the
promoter (e.g. from Hbb-b1) but no other parts of
the globin gene.
Greater Enhancement by Individual Cis-acting Elements than the HS2
Core in MEL Cells--
The HS2 enhancer contains multiple cis-acting
sequences, and thus, the reduced enhancement by HS2 in MEL cells could
result from less activity from specific positive cis-acting elements, greater activity from negative cis-acting elements, or both. Therefore, we compared the activity of multimers of individual transcription factor binding sites with that of the entire enhancer in both cell
lines. Previous observations (26, 27, 31, 32) showed that in K562 cells
the tandem MAREs are not sufficient for full enhancement. A similar
result is obtained using the cationic lipid as the transfection reagent
for K562 cells, assayed at increasing amounts of transfecting DNA (Fig.
2A). A second cis-element of HS2 known to contribute to enhancement in K562 cells is the E box at
position 8701 in the sequence file HUMHBB (32). We tested the ability
of a multimerized E box sequence, containing five copies, to enhance
independently of other elements in HS2 (Fig. 2B). The
pentamer of the E box enhanced 3-fold, which is significant (p < 0.05, Table I) but
much less than the enhancement by the HS2 core.

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Fig. 2.
Enhancement from individual cis-acting
elements compared with the core of HS2. A and
B, in K562 cells expression from luc and HS2 is
compared with that from luc enhanced by human MARE
(hMARE) sequences (A) or a human E box multimer
(B) composed of five copies of the 8701 E box
sequence. Diagrams of the constructs are shown between panels
A and B. The MAREs are represented by two shaded
rectangles, and the E boxes are represented by five rectangles with
darker shading. Rectangles with lighter shading represent
some of the other conserved blocks in the HS2. Results are plotted as
luciferase activity as a function of mass of DNA used in the
transfection. C and D, in MEL cells, expression
from 106 and 106HS2 were compared with that from 106 enhanced
by human MARE sequences (C) or a human E box multimer
(D) ligated to 106. Diagrams of the constructs are shown
between panels C and D.
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In contrast, in MEL cells, the MAREs enhance the HBB
promoter more strongly than does the HS2 core (Fig. 2C).
Also, the pentamer of the E box stimulates expression of the linked
HBB promoter as strongly as the intact core does (Fig.
2D). The enhancements by both the E box multimers and the
intact HS2 core are significant (p < 0.01), and they
are not significantly different from each other. Thus the reduced
enhancement in MEL cells is not exclusively from reduced activity of
either of these transcription factor binding sites, since both the
MAREs and the E boxes were as active or more active than the intact
core. These results suggest that a cis-acting element in HS2 has a
strong, negative effect in MEL cells.
Effects of HS2 Cis-elements in MEL Cells--
To examine the
contributions to enhancement by groups of cis-elements within the HS2
core, we tested the activities of a deletion series from the 5' and 3'
ends of the HindIII-XbaI HS2 core. Members of
each series were linked to the HBE-luciferase reporter gene and transfected into MEL cells. Two different experimental protocols were followed. In the first, the amount of DNA used in the transfection was titrated to find an optimal concentration for enhancement. In the
second protocol, the transfections were repeated in triplicate at the
optimal DNA concentration so that the results could be analyzed
statistically. As shown in Fig. 3, the
terminal deletions reveal negative cis-elements in HS2.

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Fig. 3.
5' and 3' deletions of the HS2 core fragment
assayed in MEL cells. A, diagrams of a series of
progressive deletions from the 5' end of the
HindIII-XbaI fragment and another series of
deletions from the 3' end. These were ligated to luc and assayed by
transient transfection. Conserved sites between 8650 and the E box
bound by upstream stimulatory factor (E/USF) are
indicated by shaded boxes in the map of the HS2 core but are
not shown in the terminal regions (HindIII to 8650 and
E/USF to XbaI). B, the luciferase activity
after transfecting MEL cells at the 2:1 charge:mass ratio with 2 µg
of test DNA is plotted for each plasmid. Each sample was transfected in
triplicate, assayed for activity, and normalized to the amount of total
protein in the sample. The means are plotted, and the error
bars show the S.D. The fold increase over no enhancer is shown
within or adjacent to the bar for each sample. C,
the amount of luciferase expression is plotted as a function of mass of
DNA transfected into MEL cells, keeping a 2:1 charge:mass ratio.
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For the triplicate transfections shown in Fig. 3B, the
HindIII-XbaI HS2 core enhanced
HBE-luciferase expression 6.6-fold (Fig. 3B).
Deletion from the 5' end increased enhancement to 11.7-fold for the
BalI-XbaI fragment. This is a significant
increase (p < 0.01 for comparison with the intact
core; Table II). A similar result was
obtained in the DNA titration experiment shown in Fig. 3C.
In this case, the enhancement increased 4-fold by deleting the
HindIII-BalI fragment (from 2.5-fold for the
HindIII-XbaI HS2 core to 10-fold for the
BalI-XbaI fragment). Thus cis-acting elements
located between HindIII and BalI exerted a
negative effect in this assay. Additional deletions into the HS2 core
caused a progressive loss of enhancement but no deletion completely
removed activity (Fig. 3 and Table II).
Deletions from the 3' end of the HS2 core revealed a second negative
element in MEL cells. For the triplicate transfections shown in Fig.
3B, removal of the 3' fragment caused an increase in
enhancement from 6.6- to 9.5-fold; this increase is significant (p < 0.02; Table II). This result was confirmed by the
DNA titration experiment in Fig. 3C in which deletion of the
3' fragment doubled the level of enhancement, from 3- to 6.8-fold.
Similar to the results with the 5' deletion series, additional
deletions from the 3' end caused a progressive reduction in enhancement
(Fig. 3), but again, all fragments retained significant activity
(p < 0.001 for each in comparison with
-luciferase,
Table II). These data show that truncation of either the 5' or 3' end
of the core of HS2 partially relieves repression in MEL cells.
The activity of a particular segment of HS2 depends on the context.
Deletion of the 5' region of HindIII to XbaI the
3' region of 8750 to XbaI shows that both have a negative
effect on enhancement when the rest of the HS2 core is present. In
contrast, both have a small but significant positive effect when
assayed by themselves. This suggests that the negative effect could
arise by interference with positive elements within the rest of HS2.
Negative Effect of HindIII-BalI Region on Both HBE and Hbb-b1 Genes
in Three Different Erythroid Cell Lines--
To examine whether the
cis-regulatory sequences at the 5' end of the HS2 core had a negative
effect only on the embryonic
-globin gene promoter in MEL cells, the
effect of its deletion was tested on promoters from the
-,
-, and
-globin genes in K562, HEL, and MEL cell lines (Fig.
4A). In this series of
experiments, the test plasmid with the globin gene promoter-driving
expression from the firefly luciferase gene was cotransfected with a
control for transfection efficiency, which was the Renilla
luciferase gene expressed from a tk promoter. The results
are plotted as a ratio of firefly luciferase activity to
Renilla luciferase activity.

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Fig. 4.
Comparison of the effects of deleting the
HindIII to BalI fragment of HS2 on
three different globin genes in three different cell lines.
A, diagram of DNA fragments within or containing HS2, which
were ligated to luciferase reporter genes driven by promoters from
HBE, HBG1, or Hbb-b1. Conserved blocks
in the HS2 core are indicated by the shaded rectangles; the
darker-shaded rectangles are the MAREs (labeled)
and E box at 8701. B, the graphs compare the expression from
plasmids containing various combinations of promoter and enhancers
after transfection of K562, HEL, or MEL cells. Expression is plotted as
the ratio between the activity of firefly luciferase (FF
luc) encoded in the test plasmids and Renilla
luciferase (R luc) encoded in a cotransfection control. All
transfections were in triplicate; the mean is plotted, and the
error bars show the S.D. The firefly luciferase in the
HBG1 constructs is from pGL3Basic (Promega), which encodes a
luciferase enzyme with higher activity and, thus, results in higher RLU
output than do the pGL2-derived firefly luciferases in the
HBE and Hbb-b1 constructs.
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As shown in Fig. 4B, removal of the
HindIII-BalI fragment doubled the level of
enhancement in K562 for both the HBE promoter (from 6-fold
in its presence to 13-fold in its absence) and the Hbb-b1
promoter (from 8- to 15-fold). However, no difference in luciferase
expression from the HBG1 promoter was measurable when the
HindIII-BalI region was removed. Interestingly,
in K562 cells, the 275-base pair BalI-XbaI
fragment worked equally as well as the 1.4-kilobase
KpnI-BglII fragment containing HS2.
HEL cells produce mainly
-globin and a small amount of
-globin,
but no
-globin, from its endogenous genes (54). Results of transient
transfections (Fig. 4B) show that the
HindIII-XbaI fragment comprising the HS2
core had almost no effect on either the HBE or
Hbb-b1 promoters. However, removal of the
HindIII-BalI negative element increased
enhancement substantially, to 3.6-fold for HBE and 5.0-fold
for Hbb-b1. In contrast to the results with K562 cells, the
HBG1 promoter responded more strongly to the
BalI-XbaI fragment of HS2 (lacking the negative
element) than to the core HindIII-XbaI fragment
(3.1- and 2-fold enhancement, respectively, Fig. 4B). This
small but significant (p < 0.01) difference indicates that the negative element can decrease enhancement of any of the three
promoters tested, although its effect is stronger on HBE and
Hbb-b1 than on HBG1.
Results of transfections in MEL cells were similar to those in HEL
cells for all three promoters (Fig. 4B). Little to no
enhancement of the Hbb-b1 or HBE promoters was
observed for plasmids containing the HS2 core
(HindIII-XbaI fragment), but both promoters
showed substantial enhancement with the BalI-XbaI
fragment of HS2 (5.8-fold for HBE and 7.0-fold for
Hbb-b1). In contrast, the HBG1 promoter was
enhanced by the HS2 core (3.5-fold), but even this increased to
8.8-fold upon deletion of the negative element. Thus the 5' HindIII-BalI fragment is exerting a negative
effect on all three promoters in MEL cells, with its strongest effect
on HBE and Hbb-b1.
A striking decline in enhancement is observed in MEL cells for the
KpnI-BglII fragment containing HS2 and the
HBG1 promoter (Fig. 4B, plasmid
KB).
Transfection of both K562 and HEL cells with this same plasmid DNA
showed strong enhancement by the KpnI-BglII fragment. Further investigation would be required to determine whether
this corresponds to a difference in species specificity, promoter
preference, or stage of differentiation.
Assays described so far test human HS2 on promoters of globin genes
from other species, i.e. rabbit and mouse. To test whether the human HS2 core works more effectively with a human HBB
promoter, MEL cells were transfected with constructs containing an
HBB promoter in place of the Hbb-b1
promoter. In this plasmid, the HS2 core was unable to enhance
expression from the human HBB promoter, but the
BalI-XbaI fragment still conferred 3.8-fold
enhancement (Fig. 5;
hu
HX compared with
hu
BX). These results are similar to the
results seen with the mouse Hbb-b1 promoter,
showing that the reduced enhancement is not an artifact of using a
heterologous promoter.

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Fig. 5.
Negative effect of the Human
HindIII to XbaI fragment on reporter
genes carrying the human -globin
promoter. A, MEL cells were transiently transfected
with the HBB promoter-luciferase reporter fusion carrying either no
enhancer (hu ), the (hu HX), or BalI-XbaI
fragment (hu BX). Transfections were done in triplicate, and the
firefly luciferase activity (FFLUC) from each sample was
normalized to the amount Renilla luciferase activity as
described previously. B, diagram of the
HindIII-XbaI and BalI-XbaI
fragments of HS2 upstream of the HBB promoter-luciferase
reporter fusion.
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The fold enhancements should not be compared between the results in
Fig. 4 and those in previous figures because of the difference in
experimental protocol. The inclusion of a cotransfection control does
affect the level of expression from the test plasmid (65), and thus, it
could affect the magnitude of the enhancement. However, the increase in
enhancement upon removal of the HindIII to BalI fragment of HS2 is consistently seen for HBE and
Hbb-b1 promoters in all three cell lines. It is seen with or
without cotransfection controls in HEL and MEL cells (data not shown)
and when using a different control (pRSVlacZ) in K562 cells (data not shown).
In summary, the data in Figs. 4 and 5 show that the 5'
HindIII to BalI fragment of HS2 has a negative
effect upon transient transfection of three immortalized erythroid cell
lines from two different species (human and mouse), affecting four
different globin genes. Hence the effect is not species-specific nor is it unique to one promoter.
Negative Effect of HS2 HindIII to BalI Fragment in Normal Human
Adult Erythroid Cells--
To test whether the negative effect of the
5' end of HS2 could be seen in normal human cells rather than in
continuously growing cell lines, normal hAEC were isolated by culturing
peripheral blood by the procedure of Fibach et al. (55). The
hAEC were transfected with plasmids containing the
106-luciferase
reporter gene with or without DNA fragments containing segments of HS2. The results of the transient expression assays (Fig.
6) show that the HindIII to
XbaI HS2 core segment had no effect on the reporter gene,
whereas the BalI-XbaI DNA fragment boosted
expression over 7-fold. Thus the negative effect seen for the 5'
HindIII-BalI fragment is not an artifact of
transfecting immortalized cell lines.

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Fig. 6.
Negative effect of the
HindIII to BalI region in human adult
erythroid cells. A, hAEC cells were transfected in
triplicate with 106, 106HS2 (HindIII-XbaI),
and BX (BalI-XbaI) then harvested after 2 days, and the amount of luciferase activity was assayed. The means are
plotted, and the S.D. shown as error bars. B,
diagrams of the plasmid constructs are shown in the lower portion of
the figure.
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Negative Effect of HS2 HindIII to BalI Fragment on Stable
Expression after Integration into MEL Cells--
The effect of the
5' end of the HS2 core was tested
on stably integrated DNA constructs in MEL cells. As shown in Fig.
7A and Table III, in three
independently generated pools of stably transfected MEL cells, the
BalI-XbaI DNA fragment from HS2 enhanced expression of the
106-luciferase reporter gene much more than did
the HindIII to XbaI HS2 core segment. Although
the fold enhancement for each HS2-containing construct varied among the
three pools, the BalI-XbaI fragment consistently
produced a substantially greater enhancement than did the HS2 core
fragment. Thus the negative effect of the 5'
HindIII-BalI fragment is seen on templates
integrated into the mouse chromosomes.

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Fig. 7.
Negative effect of the
HindIII to BalI region in stably
transfected MEL cells before and after induction. MEL cells were
transfected 106, HX (containing the 106 promoter and the
HindIII-XbaI core of HS2), and BX (containing
the 106 promoter and the BalI-XbaI fragment of
HS2) along with a neomycin phosphotransferase expression vector as a
selectable cotransfection marker. Pools of drug-resistant cells
containing multiple clones were assayed for the amount of luciferase
activity in triplicate. The means are plotted, and the S.D. are shown
as error bars. A, results for three sets of
stably transfected pools of cells assayed without induction. Pool 1 was
the initial pool of stably transfected cells, and pools 2 and 3 were
additional ones generated in a second, independent experiment.
B, pools 2 and 3 of stably transfected cells were
subsequently grown in the absence and presence of the inducer HMBA for
5 days and assayed for luciferase activity (triplicate assays for each
pool). The fold enhancement (luciferase activity for the indicated
construct divided by the luciferase activity for 106 under the same
condition, i.e. induced or uninduced) is shown at the top of
each bar.
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Table III
Negative effect of the 5' end of the HS2 core in stably transfected MEL
cells and in human adult erythroid cells
NA, not applicable.
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Effects of Induction of MEL Cells--
We used these pools of
stably transfected MEL cells to test whether induction by HMBA could
overcome the negative effect of the HindIII-BalI
fragment. As shown in Fig. 7B and Table III, expression of
the
106-luciferase reporter gene increased in each of two pools of
transfected cells in response to the inducer, HMBA. However, the amount
of induction was similar for each construct (almost all within a range
of 5-10 -fold) in the absence or presence of a DNA fragment containing
HS2. Thus, for these constructs, the major cis-acting sequence
affecting induction appears to be the Hbb-b1 promoter. In
particular, induction by HMBA did not overcome the negative effect of
the HindIII-BalI fragment in HS2.
Deletion Series through the HindIII to BalI Region of the HS2
Core--
The 5' negative element was mapped more precisely using
additional deletions at a finer resolution. As shown in Fig.
8, the 5' end of each deletion
corresponds to one of several sequences in the HS2 core that are
conserved among mammalian globin loci (4). MEL cells were transfected
with the series of constructs that contained no enhancer (
106), the
HS2 core (
HX), or subfragments of the HS2 core beginning at
conserved region 2, 3, or 4 (
CR2, -3, or -4, respectively) or the
BalI-XbaI region (
BX). All fragments had the
same 3' end, the SacI site in the polylinker downstream of
the HS2 core. The HS2 core conferred no enhancement on the expression
of the Hbb-b1 promoter, but CR2-, -3, and -4 constructs all
showed a substantial increase of 6.1-, 4.9-, and 5.9-fold (respectively) over the enhancerless construct
106. This is similar to the 5.2-fold enhancement obtained with the
BalI-XbaI fragment. Thus the sequence responsible
for the negative effect on HS2 enhancement is localized to a region of
20 nucleotides at the 5' end of HS2. Two conserved elements that fall
within this area are candidates for the negative cis-regulatory
activity, one at the HindIII restriction site and one in a
neighboring T-rich motif.

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Fig. 8.
Deletion series of the 5' region of the HS2
core between HindIII and BalI.
A, multiple sequence alignment of the HS2 core from 4 orders
of mammals. Matches with the human sequence are shown as
periods, and mismatches are shown as the nonmatching
nucleotide. Boxes are drawn around conserved blocks that
contain at least six gap-free columns with no more than one mismatch.
Lines below the alignment indicate the remaining DNA in each
deletion construct from the HindIII site or a 5' primer site
at CR2, CR3, or CR4 to the downstream XbaI site.
B, the mean firefly luciferase activity (Ffluc)
activity per Renilla luciferase activity for each
transfection into MEL cells is plotted. The S.D. is shown by the
error bars.
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DISCUSSION |
Many studies show that HS2 of the
-globin LCR strongly
stimulates the expression of linked globin genes in transgenic mice (17, 26, 33, 66), in stably transfected MEL (21, 26) and K562 cells
(27, 67), and in transiently transfected K562 cells (29, 30). Multiple
cis-acting sequences in HS2 contribute to and modulate the enhancement
activity (26, 32, 33, 36, 47, 49, 68). Our studies reported here show
that a 20-base pair region within the HindIII to
BalI fragment at the 5' end of the HS2 core exerts a
negative effect, whereas the remaining segment of HS2 strongly
stimulates expression.
The negative effect was observed in four different types of erythroid
cells, including three cell lines (MEL, HEL, and K562) as well as
normal hAEC. All of these cells are derived from adults, but HEL and
K562 cells are not fully committed to the erythroid lineage and express
globin genes that are maximally expressed in fetal and embryonic
development. This indicates that the negative effect of the
HindIII-BalI fragment can be exerted at many
stages of adult erythroid differentiation (e.g. before and
after commitment and at various stages of erythroid maturation).
Although developmental specificity cannot be definitively addressed
with these transfection experiments, we note that a globin gene
expressed predominately during primitive erythropoiesis (rabbit
HBE) and one expressed during definitive erythropoiesis
(mouse Hbb-b1) responded strongly to this negative element.
This was seen both in cells expressing the endogenous homolog as well
as in cells in which the homolog is silent (e.g. the effect
was seen on Hbb-b1 in MEL cells, where the endogenous gene
is expressed, and in HEL and K562 cells, where the homologous gene
HBB is not expressed). Thus the HS2 negative element is
active on genes expressed at different stages of development. This is
consistent with studies of the human HS2 in transgenic mice; both
gain-of-function (19, 20) and loss-of-function (15, 69) experiments
show a comparable effect of HS2 at all developmental stages.
Human HBG1, which is expressed predominately in fetal life,
also responded to this negative element in HEL and MEL cells, albeit
less dramatically than the other genes tested. Indeed, transfection of
a HBG1 promoter-luciferase reporter in K562 cells showed no
effect of the HS2 HindIII-BalI fragment. The
absence of an effect in K562 cells and the rather modest effect in MEL cells helps explain why this effect was not reported previously, since
the HBG1 promoter has been commonly used in transfections of
these cells (27, 30, 47). Furthermore, the HBE gene also is
frequently used in transfections of K562 cells (28, 56, 64, 65, 70),
and its expression is enhanced by the
HindIII-XbaI core of HS2, although the
enhancement is increased after deletion of the
HindIII-BalI negative element. The observation
that the HindIII-BalI fragment of HS2 has only a
modest, and sometimes no, effect on HBG1 in the same cells
where it has a strong effect on HBE and Hbb-b1
shows that this part of HS2 has some promoter specificity.
The HindIII-BalI fragment of HS2 decreases
enhancement after stable integration into MEL cell chromosomes as well
as in transiently transfected cells. The unintegrated DNA in
transiently transfected cells is readily packaged into nucleosomes (71,
72), and thus, the effects of enhancers observed in transiently
transfected cells do not result from proteins interacting with naked
DNA. However, in these experiments, it is not clear that all templates
are in the same chromatin structure. We examined pools of stably
transfected MEL cells to test the effects of different segments of the
HS2 core on templates integrated into chromosomes, and hence fully packaged into chromatin. Pools were tested so that many different integration sites could be assayed at once. These experiments showed
that the negative effect of the HindIII-BalI
fragment at the 5' end of the HS2 core is exerted even when the
template is in chromatin.
Changes in the level of enhancement conferred by fragments of the HS2
core support the results of studies where transcription factor binding
sites were specifically targeted by point mutation. For instance,
mutations in the 8701 E box reduce HS2 enhancement of the human
HBE promoter in K562 cells (32). Multimers of this same E
box sequence confer a 3-fold increase in the level of expression from
the mouse Hbb-b1 promoter in MEL cells (Fig. 2C).
Deletion of the 8701 E box (plus GATA motifs and other sequences)
decreases the level of enhancement of HS2 (compare 8701-XbaI
versus 8762-XbaI fragment in Fig. 3). This is
consistent with previous mutagenesis results. However, a deletion of
same region from the 3' end of HS2 had no effect on enhancement. One
explanation is that the presence of MAREs in the
HindIII-8750 and HindIII-CAC fragments may
influence the ability to see an effect of E boxes in the 3' deletion
series. Thus the effect observed for cis-elements is dependent on their
context, i.e. other binding sites.
Interestingly, the negative effect of the 5' end of HS2 has been
recorded in previous experiments with transgenic mice but interpreted
differently. Liu et al. (36) compared the activity of the
HindIII-XbaI HS2 core to that of a
BalI-SnaBI fragment, which has the same 5' end
but extends 49 base pairs further 3' than the
BalI-XbaI HS2 fragment used in our experiments.
The fragment lacking the HindIII-BalI fragment
had a 3-fold higher average activity. The authors suggested that this
may result from an undocumented mosaicism in some of the mouse lines.
Also, Talbot et al. (26) assayed a number of HS2 segments
for enhancement of the Hbb-b1 gene in stably transfected MEL
cells. A HaeIII-XbaI fragment (of similar end
points to the BalI-XbaI fragment discussed here)
produced a slightly greater level of enhancement than did the
HindIII-XbaI fragment in several pools of clones.
Although these differences were not considered significant in the
Talbot et al. (26) paper, they are consistent with our
demonstration of a negative element at the 5' end of HS2.
Although our studies utilizing fragments of the LCR in proximity to
various globin gene promoters are useful for dissecting regulatory
components, they leave open a large number of possibilities for how
these components could function during physiological regulation. For
instance, the negative function of the
HindIII-BalI fragment could be utilized to help
keep globin genes silent in nonerythroid cells, to turn them off in
erythroid cells at appropriate stages, or to attenuate the activation
by HS2 early in erythroid differentiation. The situation is further
complicated by the fact that multiple HSs function together in the LCR,
and our experiments do not address how this part of HS2 functions in
this context. However, it is important to know that this segment can
play a negative role.
A potential practical application of the identification of negative
elements in the LCR HSs may be found in improving vectors for globin
gene therapy. Inclusion of LCR HSs in expression constructs, including
retroviral vectors, can greatly increase the level of expression of the
target globin gene (21, 25, 73, 74), but the set of DNA fragments
needed for optimal expression has not yet been defined (4). Our
characterization of negative elements within the conventional HS2 and
the indication that they also may be operative in transgenic mice (36)
raise the possibility that re-engineering LCR constructs to remove all
such negative regions could generate an even more potent enhancer of expression.