From St. Jude Children's Research Hospital, Memphis, Tennessee and the § Bone Marrow Research Laboratory, Royal Melbourne Hospital, Parkville, Australia 3050
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ABSTRACT |
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High level expression of many eukaryotic genes is
achieved through the action of distal regulatory sequences or
enhancers. We have utilized the interaction between the
erythroid-specific enhancer in hypersensitivity site 2 (HS2) of the
human -globin locus control region and the globin gene promoters as
a model to elucidate the mechanisms governing promoter/enhancer
interactions. HS2 contains a 400-base pair core element consisting of
tandem AP1/NF-E2 motifs flanked by binding sites for multiple
ubiquitous and erythroid-specific factors. We have compared the
enhancer activity of this core element with a synthetic enhancer
lacking the factor binding sites flanking the AP1/NF-E2 motif
(HS2M). In fetal/erythroid K562 cells, enhancement of
a linked
-promoter was significantly greater with wild-type HS2 than
with HS2M. In contrast, the increase in
-promoter
activity in these cells was equivalent with either enhancer fragment.
Truncation of the binding site for the fetal/erythroid-specific stage
selector protein in the
-promoter abolished the additional enhancer
activity of HS2. Similarly, insertion of the stage selector protein
site into the
-promoter boosted enhancer activity observed with HS2
but not HS2M. In adult erythroid MEL cells, enhancement of
a linked
-promoter was significantly greater with HS2 than with
HS2M. This effect was dependent on the binding of the adult
stage-specific factor, erythroid Kruppel-like factor, to the
-promoter. Taken together, this data suggests that the
stage-specific factors binding the proximal globin promoters and the
factors flanking the AP1/NF-E2 motif of HS2 act in synergy.
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INTRODUCTION |
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Transcriptional regulation of eukaryotic genes is dependent on the
formation of multiprotein complexes on critical cis-acting regulatory sequences. Interactions between these proteins can provide
tissue and stage-specificity, potential for regulation, and high
expression levels (1, 2). Complexes assembled on adjacent DNA sites
exert their influence through cooperative binding and/or
transcriptional synergy (3, 4). In proximal promoter regions, activator
proteins are thought to increase the rate of preinitiation complex
formation through interactions with their cognate TATA-binding
protein-associated factors or other components of the initiation
complex (5-11). In this setting, the position and arrangement of the
factor binding sites is critical for transcriptional activation. The
spatial arrangement of factor binding sites within distal enhancer
elements is also critical for their function. This is evident in the
human interferon- gene enhancer and the T cell receptor-
enhancer
in which the binding of "architectural proteins" such as LEF-1 and
HMG I(Y) induce a change in DNA conformation, allowing the formation of
nucleoprotein complexes or enhanceosomes (12, 13). The coordinate
action of these architectural proteins and transcriptional activators
such as NF
B or ATF-2/c-Jun is essential for optimal enhancer
function.
In contrast to this, relatively little is known about the synergistic interactions observed between complexes binding to non-adjacent DNA sites, typified by promoter/enhancer interactions. One current model proposes that initially, DNA looping or bending is required to juxtapose the two elements with subsequent transcriptional activation depending on the efficient recruitment of the enhancer by its preferred promoter (14). This process may be mediated through direct interaction between an enhancer binding protein(s) and the polymerase complex or indirectly through another protein bound to the proximal promoter (15-17). Implicit to this and other models of enhancer action is the need for constraint where only the appropriate gene and not adjacent genes will be activated. This is particularly important in the context of intergenic enhancers, which are capable of activating multiple promoters but opt to activate only one at a given developmental stage. This process may be mediated by the presence of domain elements that demarcate functional units or by promoter selectivity (18-21).
The human -globin cluster provides an ideal model for the study of
enhancer/promoter interactions in the context of a multigene locus.
High level expression from the genes in this locus is dependent on a
region containing four erythroid cell-specific and developmentally stable DNase I hypersensitivity sites
(HS1 1-4), the locus control
region (LCR) (22-24). One of these sites, HS2, contains a powerful
erythroid-specific enhancer active in transient transfections, stable
cell lines, and transgenic mice (25-28). The core of this enhancer
consists of tandem binding sites for members of the AP1 family of
proteins, including the erythroid-specific factor NF-E2, flanked by
consensus sites for multiple ubiquitous and erythroid-specific proteins
(29-33).
High level developmentally specific expression of the individual globin
genes requires the preferential recruitment of the enhancing sequences
of the LCR to their respective promoters (34). We have previously shown
that the preferential interaction of HS2 with the -globin gene
promoter in fetal/erythroid K562 cells is mediated through the binding
of a fetal and erythroid-specific protein complex, the stage selector
protein (SSP). This complex is assembled on a sequence immediately
adjacent to the TATA box, the stage selector element (SSE) (35, 36). A
second stage-specific factor, erythroid Kruppel-like factor (EKLF) is
essential for the preferential expression of the
-globin gene in
adult erythropoiesis (37). Mice lacking this protein die in
utero of a
-thalassemic-like illness (38, 39).
In the studies reported here, we explore the mechanisms governing
enhancer/promoter interactions using HS2 and the - and
-globin
gene promoters as a paradigm. Our findings reveal that maximal enhancer
activity of HS2 in fetal/erythroid K562 cells is dependent on the
presence of the SSP binding site in the proximal
-promoter.
Similarly, EKLF appears integral for the full enhancement of
-promoter activity by HS2. Interestingly, these effects appear to
depend upon synergistic transcriptional activation between the promoter
bound stage-specific proteins and factor binding sites flanking the
tandem AP1/NF-E2 element of the enhancer.
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EXPERIMENTAL PROCEDURES |
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DNA Construction--
Constructs containing the -globin
promoter (
385-+38) or a hybrid
SSE promoter subcloned
5' to the coding sequence of the chloramphenicol acetyltransferase
reporter gene (
CAT,
SSECAT) and the
-promoter
(
260-+35,
53-+35, or
35-+35) subcloned 5' of the firefly
luciferase gene (
LUC,
53
LUC,
35
LUC) have been described
previously (26, 36). Constructs containing the 734-bp HindIII-BglII fragment of HS2 or a synthetic HS2
(HS2M), containing the tandem NF-E2/AP-1 core enhancer
flanked by neutral DNA to ablate all previously reported co-enhancer
footprints but retaining the spacing between the core enhancer and
adjacent promoter have been described previously (40). Briefly, the
neutral DNA was designed to be devoid of LCR consensus sequences. The
5' component of the neutral DNA consisted of a 169-bp
XhoI-HindIII polymerase chain reaction fragment
of pBR322 backbone and a 189-bp BglII-SalI fragment of the ampicillin resistance gene. An additional 370-bp BglII-SalI polymerase chain reaction fragment of
the tetracycline resistance gene was utilized as the 3' component.
DNA Transfection and Transient Assays-- Plasmid DNA was prepared over double cesium chloride gradients. DNA was transfected into human (K562) or mouse (MEL) erythroleukemia cells by electroporation as described previously (36). K562 cells were then induced with 20 mM hemin and MEL cells with 2% Me2SO. After 48 h, cells were harvested and lysed with Triton X-100 for luciferase assays or repeated freeze-thaw cycles for CAT assays. Luciferase activity was assayed using the Promega system on a Monolight 2001 luminometer (Analytical Luminescence Laboratories), and linearity of the assay was confirmed with serial dilutions. Luciferase lysates had equivalent protein levels. CAT activity was determined as described previously (36) and standardized for lysate protein concentration. Constructs were only compared within the same series of experiments, and the results shown represent at least six different transfections with two independent plasmid preparations. Statistics were calculated using Student's t test; values of p < 0.05 were considered significant.
Production of Stable Cell Lines--
K562 erythroleukemia cells
(2 × 107) were transfected with 10 µg of
appropriate -promoter vector DNA and 1 µg of pCINeo (Promega). Cells were selected in G418 (500 µg/ml) for 21 days. Multiple pools
were analyzed for luciferase reporter gene expression after 48 h
of incubation with hemin. To establish a K562 cell line overexpressing EKLF, these cells were transfected with the eukaryotic expression vector pCIFLAGEKLF, which contains the complete murine cDNA tagged with the FLAG epitope under the control of the viral cytomegalovirus promoter. Cells were selected in G418 (500 µg/ml), and clones with
the highest level of transgene expression were identified by Western
blotting utilizing a specific anti-FLAG antibody and the ECL
chemiluminescence system (Amersham).
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RESULTS |
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HS2 Enhancer Activity in K562 Cells--
The erythroid-specific
enhancer in HS2 has been mapped to a 46-bp element containing a tandem
binding site for the AP1 family of proteins (41). Adjacent to this
element (5' and 3') are multiple consensus sites for erythroid-specific
and ubiquitous transcription factors (YY1, USF, SCL, GATA-1, Sp1) (Fig.
1) (30-33). We initially examined the
ability of the AP1/NF-E2 binding sites alone or in the presence of the
adjacent factor binding sites to enhance expression from the - or
-promoter in K562 cells, a model of the fetal erythroid environment
(42). To achieve this we compared the 734-bp
HindIII-BglII fragment of HS2, which contained
the AP1/NF-E2 element and adjacent sites, to a synthetic HS2
(HS2M), which contained the 46-bp element flanked by
neutral DNA to retain the spacing between the enhancer and adjacent
promoter. Each HS2 fragment was linked to a
260
-promoter/luciferase or a
378
-promoter/CAT hybrid gene and
electroporated into K562 cells. Enhancerless promoter/reporter hybrids
served as controls. As shown in Fig. 2,
the addition of the 46-bp AP1/NF-E2 element alone to either the
- or
-promoter containing construct augmented expression 6-fold
(constructs 1 and 2; constructs 4 and 5). This finding is consistent
with other studies which have shown that partial enhancer activity is
observed in the context of 46-bp element alone (29, 30). Interestingly,
linking the wild-type HS2 to the
-promoter construct resulted in an
additional 4-fold increase in transcriptional activity to that observed
with HS2M (constructs 2 and 3) (p < 0.05).
In contrast, no further increase in
-promoter activity was seen with
wild-type HS2, despite the presence of the multiple transcriptional
activator sites flanking the 46-bp AP1/NF-E2 element (constructs 5 and
6).
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Maximal Enhancer Activity of HS2 Is Dependent on Specific Sequences
in the Linked Promoter--
The preferential interaction of HS2 with
the -promoter in K562 cells is mediated, in part, through binding of
the SSP to the SSE (35, 36). We therefore postulated that the
difference in maximal enhancer activity observed with HS2 and
HS2M may also be dependent on the presence of a specific
binding site in the promoter. To examine this, we evaluated the effect
of truncation mutants of the
-promoter on the ability of HS2 or
HS2M to enhance expression. As seen in Fig.
3A (constructs 1 and 2), the
differential activity between HS2 and HS2M was retained
with truncation of the
-promoter to nucleotide position
53
relative to the CAP site (p < 0.001). In contrast, deletion of the SSE (between
53 and
35) resulted in diminution of
the enhancer activity of the wild-type HS2 to levels not statistically significantly different to those observed with HS2M
(construct 3 and 4). In fact, the reporter gene expression observed with deletion of either the SSE or the sites flanking the AP1/NF-E2 element, or both, was not statistically different (compare construct 2, 3, and 4).
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Multiple Binding Sites Flanking the AP1/NF-E2 Element Are Required
for Maximal Enhancer Activity--
To determine whether our previous
findings were secondary to a direct interaction between an HS2 binding
protein and the SSP, we constructed a series of scanning mutants
ablating each of the HS2 protein binding sites. These mutants were
linked to the hybrid SSE-promoter and a CAT reporter
gene and transfected into K562 cells. Transcriptional activation of the
promoter was maintained with mutation of all sites flanking the
AP1/NF-E2 element suggesting that no individual site is essential for
the synergy observed with the SSE (Table
I). In contrast, mutation of the
AP1/NF-E2 binding site substantially reduced the activity of the hybrid promoter (Table I).
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Maximal HS2 Enhancer-dependent Transcription of the
-promoter Is Dependent on Binding of EKLF--
The inability of the
binding sites flanking the AP1/NF-E2 element to augment
-promoter
activity in K562 cells coupled with the observation that these sites
are essential for high level
-gene expression in adult erythroid
cells suggested that a developmentally specific protein may also be
necessary for maximal enhancer-dependent transcription of
the
-promoter. To assess this, HS2 or HS2M was linked to
the wild-type
-promoter/CAT gene and transfected into the adult
erythroid cell line, MEL. An enhancerless
-promoter served as a
control. As seen in Fig. 6A,
reporter gene activity was 5-fold higher with HS2M than
with the promoter alone (constructs 1 and 2). In contrast to the
results in K562 cells, an approximate 5-fold enhancement was observed
in the presence of the sites flanking the AP1/NF-E2 motif (constructs 2 and 3) (p < 0.01). To determine whether the adult
stage-specific factor EKLF was essential for maximal enhancer activity,
we examined the effect of mutating the
-promoter CACCC box on the
expression of HS2
CAT and HS2M
CAT in MEL cells.
Disruption of the EKLF binding site crippled expression from both
promoters to undetectable levels (data not shown). To circumvent this
problem, we utilized the observation that significantly higher levels
of EKLF are observed in MEL cells than K562 cells (43). We examined
whether maximal HS2 enhancer-dependent transcription of a
linked
-promoter could be restored in K562 cells overexpressing EKLF
(Fig. 6B). A parental K562 cell line (EKLF
) or a line
overexpressing EKLF (EKLF+) were transfected with HS2
CAT,
HS2M
CAT, or
CAT alone (Fig. 6C).
-promoter activity in the absence of the enhancer fragment was
increased 2.5-fold in the EKLF+ line compared with the parental line
(Fig. 6C, (1)). The addition of HS2M
yielded a similar increase in
-promoter activity in both parental and EKLF-overexpressing lines (Fig. 6C, compare
(1) with (2)). In contrast, the addition of the
HS2 fragment containing sites flanking the AP1/NF-E2 element
demonstrated a marked differential effect between the two cell lines,
with an 18-fold increase in the EKLF+ line (Fig. 6C,
(3)) compared with a 4-6-fold increase in the parental line
(Fig. 6C, compare (1) with (3)).
Similar activation was observed in the context of an HS2 fragment in
which the CACCC motif had been mutated indicating that binding of EKLF to the enhancer is not required for this effect (data not shown).
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DISCUSSION |
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The studies presented here explore the mechanisms underlying
enhancer-dependent transcription of a linked promoter.
Utilizing elements in the human -globin locus as a model, we have
demonstrated that the maximal activity of HS2 is dependent on the
binding of multiple erythroid and ubiquitous factors to the enhancer.
It is also dependent on the binding of the erythroid-specific factors EKLF and SSP to the linked globin promoters. By virtue of the restricted expression pattern of these factors, maximal
enhancer-dependent activation of the
- and
-globin
promoters is confined to adult or fetal erythroid cell lines,
respectively. EKLF and SSP appear to act in synergy with the
enhancer-binding proteins flanking the AP1/NF-E2 binding site to
maximize the transcriptional activation of the globin promoters by
HS2.
Human HS2 represents an ideal element for the study of
enhancer/promoter interactions. Like many enhancers it requires
multiple binding motifs within its core to mediate positive effects on expression of linked genes in transfected cells or transgenic mice
(30-32). As seen in our studies and those of other investigators, the
46-bp element containing the AP1/NF-E2 binding site is essential for
enhancer function and inducibility (26, 31). However, disruption of the
sites flanking the AP1/NF-E2 element reduce enhancer function to
varying degrees suggesting that HS2 functions as a multiprotein
enhanceosome. Data from our studies, and others, indicate that no
single site outside the 46-bp element is critical for enhancer effects
as the reduction in activity seen with mutation of footprinted sites
are modest (31-33). Studies in transgenic mice and MEL cells show that
maximal enhancement of -gene expression is only observed in the
presence of all binding sites (31, 44-46). Replacement of the sites in
HS2 that flank the AP1/NF-E2 element with multiple AP1/NF-E2 sites
results in dramatic reduction in enhancer activity, suggesting that a
specific cooperativity exists between all enhancer binding proteins
(31).
The importance of promoter sequences in transcriptional activation of
the -gene by the LCR has been previously demonstrated in MEL cells.
These studies identified TATA, CACCC, and CAAT boxes as sequences
critical for LCR responsiveness. Although enhanced
-gene expression
was observed in the presence of the TATA box alone, maximal enhancement
was dependent on the presence of all three sites (47). Our findings
suggest that the binding sites for the stage-specific proteins EKLF and
SSP in their respective promoters are important for maximal enhancement
by HS2. The increase in transcriptional activation observed in the
presence of these factors cannot solely be attributed to their binding
regulatory sequences, as significant differential effects are observed
in the presence or absence of the wild-type enhancer. Truncation of the
SSE in the presence of the HS2M does not alter the level of
promoter activity and the addition of this element to the
-promoter
has no effect in the absence of the fully active HS2. Similarly, the
transcriptional activation of the
-promoter observed with EKLF
expression in K562 cells is markedly different in the presence of the
wild-type enhancer. Thus maximal enhancer activity appears to depend on
EKLF or SSP and the factors binding to sites adjacent to the AP1/NF-E2
motif.
Although the studies presented here utilize HS2, the studies of Morley et al. (28) indicate that this site alone is sufficient for developmentally specific expression of the human globin genes in transgenic mice. This effect may be due to functional homology between the sites mediated through the conservation of topology of protein binding sites in all human HS and in the corresponding HS in mouse, chicken, and rabbit (48-50). Our observations with HS2 may therefore be representative of the interactions between the globin gene promoters and the complete LCR.
Despite the fact that HS2 is situated immediately adjacent to the globin promoters in these and other studies, it appears that this element does not simply function as an extension of the promoter in this context (31). This is most evident in constructs in which the AP1/NF-E2 site has been mutated and transcriptional activation by the entire enhancer is lost. Similarly, replacement of sites flanking the AP1/NF-E2 motif with alternate transcriptional activator sites significantly reduces promoter enhancement (31). The effects of the sites flanking the AP1/NF-E2 motif do not appear to be secondary to the topology of the plasmids as results with supercoiled DNA were identical to those observed with linearized plasmids (data not shown). Similarly, the presence of flanking chromatin did not influence cooperative effects of the enhancer bound proteins and EKLF or SSP as stable transfectants reproduced the results we observed in the transient assays (Fig. 3B and data not shown).
The mechanisms governing the recruitment of multiprotein enhanceosomes
to individual promoters remains unclear. We have recently demonstrated
a protein/protein interaction between NF-E2 and hTAFII130, which allows juxtaposition of the LCR and proximal globin promoters (51). However, we have been unable to define a similar direct interaction between enhancer-bound proteins outside the AP1/NF-E2 motif
and EKLF or SSP, suggesting that the stage-specific transcriptional activation observed with these factors is secondary to a separate mechanism.2 An alternate
hypothesis to explain our findings is that binding of the
stage-specific factors to their cognate sites increases the
accessibility of the enhancer-bound factors to the transcriptional machinery assembled on the promoter. In support of this is our recent
evidence that EKLF interacts with cAMP response element-binding protein, a co-activator putatively involved in chromatin
remodeling.3 Interestingly,
mice nullizygous for EKLF demonstrate loss of the hypersensitivity site
that forms on the -promoter during definitive erythropoiesis. The
EKLF/cAMP response element-binding protein interaction may result in
displacement of core histones allowing recruitment of the basal
transcriptional machinery and subsequent interaction between this
complex and enhanceosome-bound proteins.
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ACKNOWLEDGEMENTS |
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We thank A. W. Nienhuis for continuing support and helpful comments on the manuscript. We thank Jim Bieker for the EKLF cDNA and N. Tran and A. Becher for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by the National Health and Medical Research Council of Australia, the Wellcome Trust (to S. M. J.), National Institutes of Health Grant PO1 HL53749-03, Cancer Center Support CORE Grant P30 CA 21765, an ASH Junior Faculty Scholars Award (to J. M. C.), and the American Lebanese Associated Charities (ALSAC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Joint senior authors of this work.
¶ To whom correspondence should be addressed.
1 The abbreviations used are: HS, hypersensitivity site; LCR, locus control region; SSP, stage selector protein; SSE, stage selector element; EKLF, erythroid Kruppel-like factor; bp, base pair(s); CAT, chloramphenicol acetyltransferase.
2 J. M. Cunningham and S. M. Jane, unpublished material.
3 S. Pattinson, S. M. Jane, and J. M. Cunningham, unpublished material.
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REFERENCES |
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