From the
Interaction between the stage selector element (SSE) in the
proximal
The normal developmental program of the human
The concept
of competition between globin gene promoters for a shared enhancer as a
mechanism of developmental regulation was originally proposed for the
chicken globin locus. In this system, silencing of the embryonic
Although numerous protein footprints have been
demonstrated in HS2, the site of interaction with the SSE is unknown.
HS2 contains a core enhancer element that binds the transcription
factors NF-E2/AP-1, LCRF-1, and
NRF-2
(17, 18, 19) . Additional cis-acting
elements located 5` and 3` to the core enhancer have also been defined.
These sites, known as co-enhancers, bind the transcriptional regulators
Sp1, GATA-1, USF, and YY1 and contribute to high level expression in
stable transfectants and transgenic mice
(20, 21) . In
the studies delineated here, we sought to determine whether the core
enhancer of HS2 was sufficient in isolation for the preferential
interaction between HS2 and the
Although the promoter elements of the human globin genes have
been exhaustively studied, little is known about the role of the
untranslated leader sequences of these genes. We now report the
presence of two novel elements in the 5`-UTR of the
We have
previously shown that the competitive advantage of the
Our
studies lead us to propose a model whereby the 2 stage selector
elements function by stabilizing the interaction between looping
upstream regulatory sequences of the LCR and the transcriptional
machinery of the
The co-incident derepression
of
The function of the 5`-UTR has been
demonstrated in viral and eukaryotic genes as centering predominantly
on translational control (32-35). However, transcriptional
activity of this region has also been demonstrated
(36) .
Sequences downstream of the HIV-1 and HIV-2 promoters have been shown
to influence basal transcription secondary to binding of the
transcription factor LBP-1
(37) . In eukaryotes, the
Drosophila heat shock gene, hsp22, is
transcriptionally regulated by a small region between the cap site and
nucleotide +14
(38) . Similarly, the human gastrin gene
appears to be transcriptionally responsive to the leader
sequence
(39) . The role of the 5`-UTR in globin gene regulation
remains virtually unexplored. The transcriptionally active sequences
reported here suggest that regulation of the
We thank Merlin Crossley and Stuart Orkin for the gift
of anti-GATA-1 antibody and nuclear extract from COS cells
overexpressing GATA-1.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-globin promoter and hypersensitivity site 2 in the locus
control region partly mediates the competitive silencing of the
-globin promoter in the fetal developmental stage. We have now
demonstrated that a second SSE-like element in the 5`-untranslated
region of the
-gene also contributes to this competitive silencing
of the
-gene. Utilizing transient transfection assays in the fetal
erythroid cell line, K562, we have shown that the core enhancer of
hypersensitivity site 2 can preferentially interact with the proximal
-promoter in the absence of the SSE, completely silencing a linked
-promoter. Mutation of a 20-base pair sequence of the
-gene
5`-untranslated region (UTR) led to derepression of
-promoter
activity. A marked activation of
-promoter activity was also
observed with this mutation, suggesting the presence of a repressor.
Fine mutagenesis dissected these activities to different regions of the
5`-UTR. The stage selector activity was localized to a region centered
on nucleotides +13 to +15. Electromobility shift assays
utilizing this sequence demonstrated binding of a fetal and
erythroid-specific protein. The repressor activity of the 5`-UTR was
localized to tandem GATA-like sites, which appear to bind a complex of
two proteins, one of which is the erythroid transcription factor
GATA-1. These results indicate that the 5`-UTR of the
-gene
contains sequences that may be important for its transcriptional and
developmental regulation.
-globin locus
is characterized by two switches in gene expression: from embryonic
(
) to fetal (
and
) beginning in
the 5th week of gestation and from fetal to adult (
and
)
during the perinatal period. This developmental program is governed by
a diverse array of regulatory mechanisms
(1) . Cis-acting
elements within or immediately flanking the genes themselves can confer
tissue and temporal specificity in transgenic mice, but the levels of
expression are low
(2, 3, 4) . High level
expression is restored when the genes are linked to the locus control
region (LCR),
(
)
a regulatory region 6-20
kilobases 5` of the
-globin gene that is characterized by four
erythroid-specific DNase 1 hypersensitivity sites (5, 6). This enhanced
expression is achieved without disruption of the temporal regulation of
the
- and
-genes, which appear to be autonomously
silenced
(7, 8) . In contrast, the increase in
-gene
expression mediated by the LCR is accompanied by expression throughout
development. Restoration of
-gene expression confined to the adult
developmental stage is only achieved when the
-gene is linked in
the normal configuration
(9, 10, 11) . These
results suggest that competition between the genes for LCR sequences is
essential for appropriate regulation of the
-gene.
-globin gene in the adult stage of erythropoiesis is mediated by a
sequence in the
-promoter, the stage selector element
(SSE)
(12) . An erythroid and developmentally specific protein,
NF-E4, appears to facilitate the preferential interaction between the
chick
-promoter and enhancer by binding to both regions and
establishing a protein bridge that brings these two elements into
apposition
(13) . In the human
-globin cluster, we have
identified an analogous SSE in the proximal
-promoter. This
sequence fosters a preferential interaction between the
-promoter
and hypersensitivity site 2 (HS2) of the LCR in the fetal stage of
erythropoiesis, thus silencing a linked
-promoter
(14, 15) . As with the chicken SSE, the
ability of this element to confer a competitive advantage to its
promoter is dependent on the binding of a developmentally specific
protein, the stage selector protein (SSP). The SSP is a heteromeric
complex consisting of a ubiquitous transcription factor, CP2, and a
fetal/erythroid-specific partner protein
(16) . CP2 has recently
been demonstrated to represent the human homologue of chicken
NF-E4
(16) .
-promoter. Our results define a
novel SSE-like element in the 5`-untranslated region (5`-UTR) of the
-globin gene, which appears to preferentially interact with this
enhancer in the presence of a developmentally specific protein and thus
contribute to the competitive silencing of the
-gene in the fetal
stage of erythropoiesis. We have, in addition, identified a separate
negative regulatory element in this region.
DNA Construction
pUC007, a pUC-based
plasmid (17), was used as the vector in all constructs. Constructs
containing the -globin promoter (-385 to +38) subcloned
5` of the coding sequence of the chloramphenicol acetyltransferase
reporter gene (
CAT) and the
-promoter (-260 to +35
or -35 to +35) subcloned 5` of the firefly luciferase gene
(
LUC or -35
LUC) have been previously
described
(14, 17) . HS2 was subcloned as a 734-bp
HindIII-BglII fragment into the
HindIII-BamHI fragment of pUC007. The
XhoI-AatII fragments of
CAT and
LUC were
subcloned 3` of HS2 into a SalI-AatII fragment
(HS2
CAT, HS2
LUC). A synthetic HS2 (HS2
)
containing the tandem NF-E2/AP-1 sites flanked by neutral DNA was
constructed by PCR. The HS2
retains the spacing between the
core enhancer and the adjacent promoter but lacks all the previously
reported co-enhancer footprints. Neutral sequences were designed to be
devoid of consensus sequences for known transactivators of the LCR.
Initially, a 163-bp XhoI-HindIII PCR product of the
pBR322 backbone and a 189-bp BglII-SalI PCR product
of the pBR322 ampicillin resistance gene were subcloned 5` and 3` to
the 46-bp enhancer core in pUC007
(22) . An additional 370-bp
BamHI-SalI PCR product of the pBR322 tetracycline
resistance gene was subcloned 3` to retain the natural spacing. The
entire HS2
was released as a SalI-AatII
fragment and subcloned 5` of either
CAT or
LUC
(HS2
CAT, HS2
LUC) using the
XhoI/SalI-AatII strategy outlined above.
Dual promoter constructs linking the
- and
-260
-promoter (HS2
CAT
LUC,
HS2
CAT
LUC) or the
- and
-35
-promoters (HS2
CAT-35
LUC,
HS2
CAT-35
LUC) with their respective reporter
genes were obtained using the same strategy.
Oligonucleotides and Site-directed
Mutagenesis
Oligonucleotides were synthesized on an Applied
Biosystems synthesizer model 380B using phosphoramidite chemistry and
purified on Sephadex G-25 columns (Pharmacia Biotech Inc.). Prior to
use in gel mobility shift assays, complementary strands were annealed
in 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 10
mM MgCl by heating to 95 °C for 5 min and slow
cooling to room temperature. PCR site-directed mutagenesis was
performed as described
(23) . Briefly, mutants were made by PCR
in 2 halves incorporating a PstI restriction site at the
3`-end of one and the 5`-end of the other. Products were cleaved with
PstI, ligated with T4 DNA ligase, and the full-length mutant
amplified by PCR using the external primers. Once subcloned into
plasmids, the sequence was verified by the chain termination method
with Sequenase (U. S. Biochemical Corp.).
Cell Lines and Nuclear Extracts
Human
erythroleukemia (K562) cells, murine erythroleukemia (MEL), and HeLa
cells were grown in improved minimal essential medium (Biofluids) with
10% fetal calf serum and 50 µg/ml gentamicin. Nuclear extracts were
prepared as described
(14) .
DNA Transfection and Transient
Assays
Plasmid DNA was prepared over double cesium chloride
gradients. 3.5 10
K562 cells were mixed with
equimolar amounts of the test plasmids in the presence of carrier
plasmid (pUC 9) to a total of 50 µg. Electroporation was performed
with a Bio-Rad gene pulser apparatus at 960 microfarads and 0.2 kV.
After transfection, cells were grown in the presence or absence of 20
µM hemin, and each sample was split in two and harvested
after 48 h. Cell lysates from one half were prepared by repeated
freeze-thaw cycles and assayed for CAT activity as
described
(24) . The remaining half was lysed with Triton X-100,
and luciferase activity was measured on a Monolight 2001 luminometer
(Analytical Luminescence Laboratories). The assay was linear from
10
to 10
light units; samples above or below
this were reassayed with appropriate dilution. CAT activities were
standardized for lysate protein concentration. Luciferase lysates had
equivalent protein levels. In our previous studies, we had demonstrated
that inclusion of an internal control reporter plasmid was unsuitable
in dual promoter constructs
(14) . Standard single promoter
constructs were used as experimental controls, and constructs were only
compared within the same series of experiments. Results shown represent
at least six different transfections with two independent plasmid
preparations in induced cells. Similar trends were observed in separate
transfections of uninduced cells. Statistics were calculated using
Student's t test; values of p < 0.05 were
considered significant.
RNase Protection Assay
80 µg of total
RNA was prepared from K562 cells 48 h after electroporation using
RNAzol B (Tel-Test, Friendswood, TX) according to the
manufacturer's instructions. Probes were constructed by
subcloning XhoI-BamHI PCR fragments containing the
-35-wild-type or mutant promoters and the first 243 bp of
the luciferase coding sequence into the XhoI-BglII
fragment of pSP73. Sequence integrity was verified by dideoxy
sequencing. High specific activity radiolabeled transcripts were
generated with SP6 RNA polymerase. The predicted length of protected
fragments from correctly initiated transcripts was 284 bp. RNase
protection assays were performed as described
(25) , and samples
were electrophoresed on an 8% sequencing gel.
Gel Mobility Shift Assay
Assays were
performed with 10 cpm of probe added to a 20-µl
reaction containing varying amounts of nuclear extract, 500 ng of
poly[d(GC)], 6 mM MgCl
, 16.5 mM
KCl, and 100 µg of bovine serum albumin
(26, 27) .
For competition assays, a 150-fold molar excess of unlabeled
double-stranded oligonucleotide was added with the probe. In antibody
studies, 3 µl of pre-immune serum or rat or rabbit anti-mouse
GATA-1 antibody (kindly provided by Merlin Crossley and Stuart Orkin,
HHMI, Childrens' Hospital, Boston) were preincubated for 10 min
with the binding reaction prior to addition of the probe. After
incubation at 4 °C for 10 min and 25 °C for 20 min, samples
were electrophoresed on a 4% non-denaturing polyacrylamide gel in 0.5
Tris-borate-EDTA buffer for 90 min at 10 V/cm.
The Preferential Interaction between the Core
Enhancer of HS2 and the
The erythroid-specific enhancer in HS2 has been mapped
to a 46-bp fragment consisting of tandem NF-E2/AP-1 binding
sites
(17, 22) . In our initial experiments, we sought to
determine whether this minimal enhancer was sufficient to
preferentially interact with the -Promoter Is Not Dependent on the
SSE
-promoter when in competition
with the
-promoter in a fetal erythroid environment. A 734-bp
HindIII-BglII fragment of HS2 and a synthetic HS2
(HS2
) containing the 46-bp enhancer flanked by neutral DNA
to retain the spacing between the enhancer and adjacent promoter but
ablating all reported protein binding sites were compared. Initially,
each HS2 fragment was linked to the -378
-promoter with a
CAT reporter gene or the -260
-promoter with a luciferase
reporter gene and transfected into K562 cells. With the
-promoter,
reporter gene activity was 4-fold higher in the context of HS2 than
HS2
(data not shown). This presumably reflects the effect
of positive regulatory elements in HS2 outside the core, which we refer
to as co-enhancers. By contrast with the
-promoter, reporter gene
activity was equivalent with HS2 and HS2
in K562 cells
(Fig. 1, constructs1 and 4). To
determine if the core enhancer was sufficient for the competitive
advantage of the
-promoter in the fetal environment, the
-378
-promoter/CAT reporter gene and -260
-promoter/luciferase reporter gene were linked in competition for
HS2 or HS2
(HS2
CAT
LUC,
HS2
CAT
LUC) and transfected into K562 cells.
Constructs linking the HS2 fragments to the
-promoter/CAT reporter
hybrid served as controls. As seen in Fig. 1,
-promoter
activity in the dual promoter constructs was diminished 10-fold
compared with the controls with both HS2 and HS2
(p < 0.05), indicating that the minimal enhancer was sufficient to
allow preferential interaction of HS2 with the
-promoter
(constructs2 and 5). The reduction in
-promoter activity observed in construct 5 by comparison with
construct 2 is consistent with the loss of the co-enhancer effect.
Figure 1:
The minimal
-promoter can silence a linked
-promoter in competition for
the HS2 core enhancer. A, diagrammatic representation of the
constructs used for transfections into K562 cells induced with hemin.
Solidboxes represent either the 734-bp
HindIII-BglII fragment of HS2 (constructs1-3) or the HS2 core enhancer flanked by neutral
DNA to retain equivalent spacing (constructs4-6). The hatchedboxes represent
the -385
-promoter linked to the CAT gene, and the openboxes represent either the -260 (constructs2 and 5) or the -35 (constructs3 and 6)
-promoter linked to the luciferase
reporter gene. B, reporter gene activity of these constructs.
Hatchedbars represent CAT conversion, standardized
per microgram of protein; openbars represent
luciferase activity (lysates had equivalent protein concentration).
Values shown represent the mean ± S.E. of at least six separate
transfections with two different plasmid preparations. Statistical
significance was assessed using Student's t test.
To determine if the interaction between the -promoter and the
core enhancer was dependent on the SSE, we truncated the
-promoter
to -35 relative to the transcriptional start site and examined
the effect in dual promoter constructs in the presence of HS2 and
HS2
. The residual
-promoter contained the 35
nucleotides 5` to the cap site and an additional 35 nucleotides of
5`-UTR. In the context of the wild-type HS2, truncation of the
-promoter resulted in a marked decrease in its activity,
presumably due to loss of upstream regulatory elements (Fig. 1,
construct3). Loss of the SSE in this setting
resulted in derepression of the linked
-promoter (p <
0.01) (Fig. 1, construct3). However, the level
of
-promoter activity observed in this construct was significantly
less than the activity seen when the
-promoter was linked to the
same HS2 fragment in isolation (Fig. 1, construct1), suggesting that residual competitive sequences may be
present. This was confirmed with the construct linking HS2
to the
CAT/-35
LUC cassette (Fig. 1,
construct6). In this setting, the
-35
-promoter totally retained its competitive advantage, and
the linked
-promoter was suppressed. These findings led us to
postulate the existence of alternative stage selector-like elements
that interact specifically with the HS2 core enhancer within the
proximal
-promoter.
Localization of an Alternate Stage Selector Element
in the 5`-UTR of the
To further localize
the sequences in the -35-Globin Gene
-promoter responsible for its
competitive advantage in attracting the core HS2 enhancer, scanning
mutagenesis of this region was performed. The ability of mutants to
compete with a linked
-promoter for interaction with HS2
was tested in K562 cells. As seen in Fig. 2, construct2, mutation of the
-TATA box
(-35
) totally abolished
-activity. Despite
this, no derepression of the linked
-promoter was seen, implying
that the competitive advantage of the -35
-promoter was
independent of its transcriptional activity. Mutation of the sequences
between the TATA box and the CAP site decreased -35
-promoter
activity slightly but had no effect on repression of a linked
-promoter (data not shown). In contrast, mutation of the 5`-UTR
(-35
, Fig. 2, construct3) led to a 3-fold derepression of
-promoter
activity (p < 0.05), suggesting that this area contained a
competitive element capable of preferentially attracting the HS2 core
enhancer. In addition to the derepression of the
-promoter, the
20-bp 5`-UTR mutant also resulted in a concomitant 7-fold increase in
-promoter activity (p < 0.05), suggesting that this
mutation might also have disrupted a separate negative regulatory
element. To further evaluate this repressor activity, the wild-type and
mutant -35
-promoters were linked to the
HindIII-BglII fragment of HS2 as single promoter
constructs and transfected into K562 cells and the murine adult
erythroid cell line, MEL. In both cell lines, the wild-type promoter
was less active than the mutant, and a comparable degree of repression
was observed in each, indicating that this effect was not
developmentally specific (data not shown).
Figure 2:
Mutation of the 5`-untranslated region of
the minimal -promoter activates
- and derepresses linked
-promoter activity. A, diagrammatic representation of
constructs. In construct1, the wild-type minimal
-promoter (-35 to +35) has been linked to the
-385
-promoter in competition for the HS2 core enhancer.
Constructs2 and 3 are identical except for
mutations of the
-TATA box and 5`-untranslated region between
+8 and +27, respectively. B, reporter gene activity
of corresponding constructs. Hatchedbars represent
CAT conversion, and openbars represent luciferase
activity.
To evaluate the
contribution of the 5`-UTR SSE-like element to the suppression of a
linked -promoter in the context of larger
-promoter and HS2
fragments, the 20-bp 5`-UTR mutation was made in the
-260
-promoter, and the ability of this mutant to silence a
linked
-promoter in competition for the 734-bp
HindIII-BglII HS2 enhancer was tested. As shown in
Fig. 3
(construct2), with the larger HS2, loss
of the UTR SSE resulted in a significant derepression of
-promoter
activity (p < 0.05) despite the presence of an intact
-promoter SSE. As previously shown, loss of the
-promoter SSE
by truncation to -35 (construct3) resulted in
a significant derepression of linked
-activity (p <
0.05). In the absence of the
-promoter SSE, mutation of 5`-UTR
(construct4) led to a more marked derepression of
-promoter activity than that seen with construct 3 (p < 0.05). This suggests that the two SSE regions function
cooperatively. As with the core enhancer, mutation of the 5`-UTR also
led to activation of -35
-promoter activity (p <
0.05, construct4). This effect was less pronounced
in the setting of the -260
-promoter (construct2), suggesting that upstream elements in the
-promoter can compensate for the repressor activity of this
region.
Figure 3:
Mutation of the 5`-untranslated region in
the -260 -promoter also derepresses linked
-promoter
activity in the context of a larger HS2 fragment. A,
diagrammatic representation of constructs. In construct1 and 3, the wild-type -260 or
-35
-promoters have been linked to the -385
-promoter in competition for the 734-bp
HindIII-BglII fragment of HS2. Constructs2 and 4 are identical to these, respectively,
except for mutation of the
-5`-untranslated region between +8
and +27. B, reporter gene activity of corresponding
constructs. Hatchedbars represent CAT conversion,
and openbars represent luciferase
activity.
To dissect the sequences in the 5`-UTR responsible for the
SSE and repressor functions, a series of smaller mutants spanning this
area were made and tested for their ability to compete with a linked
-promoter for the HS2
enhancer. As shown in
Fig. 4
(constructs1 and 2), mutation
of the sequence from +13 to +15 (-35
)
resulted in a significant derepression of linked
-promoter
activity without reduction in
-activity (p < 0.01). No
other mutant demonstrated any
-promoter derepression (data not
shown). This suggested that the nucleotides from +13 to +15
may represent the important contact bases for a protein mediating the
SSE-like effect. It should be noted that this mutation introduced a
different sequence between +13 and +15 than the 20-bp mutant,
making it unlikely that the effect is specific to the mutant sequences.
In contrast, mutation of the 5`-UTR between +24 and +29
(Fig. 4, construct3) had no effect on linked
-promoter activity but led to a 5-fold increase in
-promoter
activity (p < 0.05). These nucleotides are centered on a
tandem inverted binding motif for the erythroid transcription factor,
GATA-1. These results suggested that the
-globin 5`-UTR contains
two distinct regulatory elements, one analogous to the SSE and another
that functions as a repressor.
Figure 4:
A
stage selector-like element and a repressor sequence are localized to
different regions of the -5`-untranslated region. A,
diagrammatic representation of the constructs used. In construct1, the -35
-promoter has been linked to the
-385
-promoter in competition for the HS2 core enhancer.
Constructs2 and 3 are identical to
construct1 except for mutations of the
5`-untranslated region between +13 and +15 or +24 and
+29, respectively. The wild-type
-5`-UTR is shown under
construct1, and mutated sequences in constructs2 and 3 are shown in lowercase. B, reporter gene activity of corresponding
constructs. Hatchedbars represent CAT conversion,
and openbars represent luciferase
activity.
Functional Mutations in the 5`-UTR Do Not Alter
Transcription Initiation
Previous studies have shown that
deletions 3` to the TATA box in the rabbit -globin promoter and
5`-UTR altered the transcription initiation
site
(28, 29) . To ensure that the effects we had
observed with the UTR mutants were not attributable to aberrant
initiation, RNase protection analysis was performed. RNA was prepared
from K562 cells transfected with single promoter constructs containing
the -260
, -35
, and -35
promoters. The full HS2 enhancer was used to increase transcript
levels. Fig. 5shows a similar pattern of one major and two minor
bands clustered around the mRNA CAP site for all three promoters. The
upper major band corresponds to the correctly initiated full-length
284-bp mRNA transcript. The minor smaller bands are consistent with the
previously reported minor transcripts and probably represent
microheterogeneity in
-globin transcription initiation
sites
(22, 28) . No new downstream initiation site was
introduced with the 5`-UTR mutation. Transcript levels with the
repressor mutant were noted to be increased in comparison to the
wild-type -35
-promoter (Fig. 5, lanes4 and 6).
Figure 5:
Mutation of the -5`-untranslated
region does not alter transcription initiation. Constructs linking the
wild-type -260 or -35
-promoters (lanes3 and 4) or the mutant -35
-promoter (lane6) were electroporated into K562 cells and RNA prepared
48 h post-transfection. RNase protection analysis was performed on 80
µg of total RNA or 10 µg of yeast RNA using 35
-wild-type (WT) or mutant (M2) RNA probes.
Probe integrity was confirmed by hybridizing to yeast RNA in the
absence of RNase (lanes2 and 5). The
arrow indicates the correctly initiated 284-bp protected
transcript.
Analysis of Proteins Binding to the
To ascertain whether the functional sequences
defined in the experiments above represented protein binding sites, we
performed electromobility shift assays (EMSA) using the wild-type
-Promoter
5`-UTR
-gene from -5 to +27 as probe (UTR 1). As shown in
Fig. 6
, three retarded bands were observed with crude K562
nuclear extract (lane1). Competition with excess
non-radiolabeled probe ablated binding activity, confirming the
specificity of these interactions (data not shown). A mutant probe (UTR
1M), which altered only the 3 bp from +13 to +15, the site of
the putative stage selector-like element, failed to bind the uppermost
of these complexes (Fig. 6, lane2). To
evaluate the tissue specificity of this complex, wild-type and mutant
probes were used to assay nuclear extracts from adult erythroid (MEL)
and non-erythroid (HeLa) cell lines (Fig. 6, lanes3-6). Binding activity of the uppermost complex was
absent in these extracts, suggesting that this complex is both fetal
and erythroid specific. Neither a highly purified preparation of SSP or
recombinant CP2 bound to the wild-type UTR 1 probe (data not shown).
Figure 6:
The alternate stage selector element binds
a protein that is fetally and erythroid restricted. A,
sequences of the upstream -5`-untranslated region probe wild-type
(UTR 1) and mutant (UTR 1M) probes used in electrophoretic mobility
shift assays. The mutation at the site of the stage selector-like
element is underlined. B, EMSA of upstream 5`-UTR
probes with crude K562, MEL, and HeLa nuclear
extracts.
To evaluate protein binding to the repressor sequence in the 5`-UTR,
a probe spanning nucleotides +10 to +42 (UTR 2) was used in
the EMSA with extract derived from K562, MEL, and HeLa cells. As seen
in Fig. 7, two retarded species were observed with all extracts.
However, the broad lower complex contained a distinct
erythroid-specific component in MEL and K562 extract (lanes1 and 2). Mutation of the tandem inverted GATA
motifs with activity in functional assays (UTR 2M) abolished binding of
both the upper ubiquitous band and the erythroid-specific band
(Fig. 7, lane9). To determine whether the
erythroid-specific band was due to binding of GATA-1, an excess of an
unlabeled probe consisting of 6 repeats of the GATA motif or anti-mouse
GATA-1 antibodies were added to the reaction mixture. As seen in
Fig. 7
, both the unlabeled competitor (lane4)
and anti-mouse GATA-1 antibody (lane6) significantly
reduced the binding of the erythroid complex without altering the
ubiquitous band. Preimmune sera nonspecifically diminished both bands
to a minor degree (lane 5). The diminution in GATA-1 binding
observed with this antisera was comparable with that observed by other
investigators.(
)
To further validate that the
erythroid complex consisted of GATA-1, nuclear extract from COS cells
transfected with a GATA-1 expression vector and extract from
untransfected COS cells was studied with the UTR 2 probe. As seen in
lanes7 and 8, the GATA-1 containing extract
produced a single retarded band, which comigrated with the
erythroid-specific band seen with K562 extract. No complex was observed
with untransfected COS extract. These results suggest that the
repressor element of the 5`-UTR binds two proteins, one of which is
GATA-1.
Figure 7:
The
repressor element of the -5`-untranslated region binds two
proteins, one of which is GATA-1. A, sequences of the
downstream
-5`-UTR wild-type probe (UTR 2), a similar probe with a
mutation at the site of the repressor element (UTR 2M), and a probe
consisting of 6 GATA repeats used as a non-radiolabeled
competitor(GATA). B, EMSA of wild-type and mutant downstream
5`-UTR probes with crude K562, MEL, and HeLa nuclear extracts
(lanes1-3 and 9) and extracts from
untransfected COS cells (lane7) or COS cells
transfected with a GATA-1 expression vector (lane8).
Competition experiments were performed with K562 extract preincubated
with non-radiolabeled GATA probe competitor (lane4),
pre-immune serum (lane5), or anti-mouse GATA-1
antibodies (lane6).
-gene, which
appear to have differing transcriptional activities. The first element,
localized to a region centered on nucleotide +14, functions as an
alternate stage selector by directing preferential interaction of the
erythroid enhancer of HS2 to the
-gene in a fetal erythroid
environment. The second element represses
-promoter activity in
both fetal and adult stages of erythropoiesis. Both of these effects
appear to be mediated by specific DNA binding proteins.
-promoter
in the fetal stage of erythropoiesis is mediated in part by the SSE in
the proximal
-promoter
(14) . We now demonstrate that a
second element in the 5`-UTR (SSE-2) also contributes to the
preferential interaction of HS2 with the
-promoter. SSE-2 is
distinct from the
-promoter SSE in that its function is unrelated
to the binding of the SSP but correlates with the binding of a second
fetal/erythroid-specific protein, which we refer to as the UTR stage
selector protein. Phylogenetic footprint analysis suggests that the
binding site for this protein is conserved in species with a distinct
fetal stage of
-gene expression
(30) . This observation is
analogous to the phylogenetic footprint data observed with the SSE,
suggesting that both factors may be integral for the competitive
silencing of the
-promoter in the fetal stage of erythropoiesis.
Examination of the binding motif of the UTR stage selector protein
reveals no obvious similarities with known consensus sequences, and
thus the identity of this factor is yet to be determined.
-promoter. SSE-2 appears to stabilize the
interaction with the core enhancer. This effect may be mediated by an
interaction between UTR stage selector protein and the enhancer protein
rather than an effect on the transcription initiation complex, since
capture of the core enhancer can occur in the absence of transcription
(Fig. 2, construct2). We have recently
demonstrated that the SSE in the proximal
-promoter may function
by recruiting co-enhancers outside the HS2 core.
(
)
In the presence of large fragments of HS2, the 2 stage
selector elements may function additively (Fig. 3). In this
context, loss of the proximal promoter SSE destabilizes the loop, and
preferential interaction with the
-promoter is lost
(14) .
However, our studies with the HS2 core enhancer suggest that loss of
the SSE in this setting induces less instability and compensation by
the 5`-UTR SSE occurs (Fig. 1). The observation that even when
both stage selector elements are removed from the
-promoter,
linked
expression does not achieve the levels seen with the
single
-promoter construct. This suggests that loss of the SSEs
negates the competitive advantage of the
-promoter, and the two
promoters now compete equally for HS2.
-promoter activity with both the 20-bp and smaller UTR mutants
suggests that a repressor element also exists in the 5`-UTR region. In
contrast to the effect of SSE-2, which must be at the level of
transcription since it affects a separate gene transcript, the
repressor effect could be either transcriptional or secondary to a
translational block or altered mRNA stability. However, we have
observed that the fold increase in
-promoter activity observed
with the mutant was mirrored by a similar increase in mRNA levels
(Fig. 5) and that the magnitude of this effect was independent of
assay time. Additional support for a transcriptional mechanism comes
from the observation that binding of the erythroid transcription factor
GATA-1 and a second ubiquitous factor was integral for repressor
activity. Although we have no evidence to differentiate which of these
factors is mediating this effect, GATA-1 has previously been
demonstrated to transcriptionally repress
-gene expression when
binding to the non-canonical (C/T)AAG motif at -117 in the
-promoter
(31) .
-gene and the
competitive balance between the
- and
-genes may be
influenced by this region and the proteins that bind to it. Although
the relative importance of these elements in the setting of the entire
cluster remains to be elucidated, the previous identification of both
the human and chicken stage selector elements in similar assays
provides precedent for their usefulness.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.