 |
INTRODUCTION |
Human
- to
-globin gene switching is coordinated by several
regulatory sequences including the gene promoters and the locus control
region (LCR).1 The LCR
contains 5 DNaseI super-hypersensitive sites (HS1-HS5) located 6 to 26 kb upstream of the
-globin gene in the
-globin gene cluster (1,
2). The
- (embryonic),
- (fetal), and
- (adult) globin genes
are positioned in the order of their temporal expression. The LCR has
enhancer activity (3, 4) and can initiate and maintain
erythroid-specific open chromatin structure to allow for the activation
of gene expression (1, 5). The HS sites of the LCR contain binding
sites for transcription factors that are critical for its activity
(reviewed in Ref. 6). In situ hybridization of primary
globin transcripts in transgenic mouse cells has provided in
vivo evidence that the LCR is likely to act as a holocomplex to
activate transcription by stably interacting with one
-like globin
gene at a time (7). A current model states that the fetal
-globin
gene and its promoter play important roles in interactions with the LCR
that result in the competitive inhibition of adult
-globin gene
expression in early development (8, 9). This is supported by the
observation that the
-globin gene is expressed inappropriately early
in mice and tissue culture cells with LCR constructs that do not
contain the
-globin gene (10-13). The factors that bind to the gene
promoters and to the LCR that are important for such interactions have
not yet been identified, although some of the DNA elements involved are
elucidated in this work.
The individual roles of the HS sites are complex and are not yet
completely understood. Studies with transgenic mice that contain
individual or combinations of HS sites, deletions of specific HS sites,
or deletions of the highly conserved core elements of the HS sites as
well as experiments with knock-out mice have provided evidence of the
roles of each HS site. HS2 and HS3 have been shown to exhibit the
majority of the enhancing activity of the LCR (14) and may participate
in additive interactions (15) or synergistic interactions for long
range enhancer activity (16).
Mice with constructs containing HS2 alone maintain correct
stage-specific expression of
- and
-globin mRNA throughout
development (10, 17) and express equivalent levels of
-globin
mRNA in the embryo and
-globin mRNA in the fetus and adult
(18). In transgenic mouse studies in which HS2 is deleted from the
intact human
-globin locus, there is only a small decrease in
expression of the
-,
-, and
-globin genes, suggesting that at
least some of the properties of HS2 may be functionally redundant (19). However, others have reported dramatic decreases in gene expression and
loss of copy-number dependence when a similar transgene is integrated
into heterochromatic regions in the mouse genome, suggesting a role for
HS2 in overcoming position effects (20). Deletion of murine HS2 in
knock-out mice resulted in normal expression of the mouse embryonic
genes but a 30% reduction of adult
-globin mRNA (21, 22). The
greater effect on the adult gene may be because of the longer distance
between the
-globin gene and the LCR or gene order. It is important
to consider that although mice are a convenient model for the
investigation of human gene expression, with respect to HS2, the mouse
-globin locus and its regulation may not be identical to that of the
human locus.
Similar studies have concentrated on the role of HS3, because HS3 is
known to demonstrate enhancing and chromatin-opening activities similar
to those of HS2 (14). In transgenic mice with constructs containing
individual HS sites, it has been shown that HS3 is the most active site
in enhancing
-globin expression during the embryonic period and also
supports
-globin expression during fetal hematopoiesis (18). In
transgenic mice containing an otherwise intact human
-globin locus,
an HS3 deletion results in a significant decrease of
-globin, an
increase of
-globin, and no change in
-globin gene expression
(19). The importance of HS3 in the human
-globin locus in overcoming
position effects has also been observed in deletion studies (20).
Deletion of murine HS3 produces alterations in the expression of the
-like globin genes similar to those caused by deletions of HS2 (21, 23). Transgenic mice with deletions of a 225-bp or a 234-bp human HS3
core have low or no detectable globin expression at all stages of
development (24) or severely reduced
-globin and no
-globin
mRNA (25). Although these results appear inconsistent with the
studies that delete the entire HS site, they substantiate the
importance of the flanking sequences of the HS sites in the activity of
the LCR. If the sequences outside of the HS core interact with the gene
promoters but lack the enhancing activity of the core element, then a
dominant-negative effect may be observed (6). This illustrates the
value of studying each HS site in the context of its core and flanking
sequences to define its role.
In previous work using transgenic mice and transfection analyses, we
have shown that the -136 to +56 region of the
-globin promoter is
necessary for competitive inhibition of
-globin gene expression in
embryonic/fetal cells. A
-globin gene with a
161 promoter can
compete successfully with the
-globin gene; therefore, an element(s)
downstream of -161 has a role in embryonic
-globin suppression (13,
26). This region of the
-globin promoter contains a CACCC element,
two CCAAT boxes, the stage selector element (SSE) and a TATA box (see
Fig. 1). Targeted gene knock-outs in mice indicate that erythroid
Kruppel-like factor (EKLF), which binds preferentially to the CACCC
element in the
-globin promoter, is responsible for positively
regulating this gene in the adult and has little direct effect on
-globin gene expression (27-29). It is plausible that an as yet
unidentified
-globin gene-specific CACCC binding factor exists and
is important for developmental switching. Simultaneous mutations of the
two CCAAT boxes in the
-globin promoter have a negligible effect on
- and
-globin gene expression in transgenic mice, so it does not
appear that these elements play an independent role in
-globin
suppression (30). Stage selector protein (SSP) binds to the 19-bp SSE
near base position
50 in the
-globin promoter. The SSE is required for competitive inhibition of
-globin expression by a
260
-globin promoter in transient transfection assays in K562 cells,
although a deletional mutation of the SSE element had little direct
effect on
-globin gene transcription in the same system (31,
32).
The current study aims to determine the roles of HS2 and HS3 from the
LCR and of the CACCC, SSE, and TATA elements in the
-globin promoter
in the suppression of
-globin gene expression early in development
using stable transfections in human HEL and K562 cells. We find that
HS3, unlike HS2, does not consistently participate in competitive
inhibition of
-globin expression by the
-globin gene. The HS3HS2
configuration has the combined enhancing activity of HS2 and HS3 and
also the developmental specificity of HS2, suggesting that HS2 and HS3
act most effectively in concert. The CACCC and TATA, but not the SSE,
elements in the
-globin promoter are involved in the inhibition of
early
-globin gene expression. This work begins to identify the
elements in the LCR and in the
-globin promoter that bind factors to
establish competitive inhibition.
 |
EXPERIMENTAL PROCEDURES |
Preparation of the Constructs for the Stable Transfection
Assays--
The constructs used in the stable transfection assays are
shown in Fig. 2 and are HS3
, HS3
, HS3HS2
, HS3HS2
,
HS3HS2
TATA
, HS3HS2
CACCC
, and HS3HS2
SSE
. Those with
HS2 contain a 1.9-kb KpnI-PvuII fragment
(GenBankTM HUMHBB coordinates 7764-9653) normally located about 11 kb
5' of the human
-globin gene. The constructs containing HS3 have a
1.9-kb HindIII fragment (HUMHBB 3266-5172). The
-globin
gene in these constructs is a 4.5-kb ApaI-EcoRV
fragment (
1250 to +3291) containing the 3' enhancer region. The
LCR
constructs contain a HindIII fragment (
1350 to
+1951) encompassing a derivative of the human A
-globin
gene, situated between the LCR sequences and the
-globin gene. The
- and
-globin genes in all of the constructs are marked by a 4-bp
insertion, made by filling in an NcoI restriction site at
position +50 using Klenow fragment to distinguish their mRNAs from
the endogenous globin mRNAs.
The mutations in the
-globin promoter TATA, CACCC, and SSE elements
were generated using the CLONTECH Transformer
Site-Directed Mutagenesis Kit. An oligonucleotide was used to mutate
the TATA, CACCC, or SSE elements and create a new restriction enzyme
site. In addition to the mutagenic primer, a second primer was used to
abolish an MluI site in the IBI-30 polylinker to facilitate the selection of mutated clones. The TATA box included in the sequence
5'-ATAAAA-3' near position -30 was mutated to a PstI restriction site (5'-CTGCAG-3'), the CACCC sequence included in the
sequence 5'-CCACCC-3' near position
140 was mutated to a ScaI site (5'-AGTACT-3'), and the sequence 5'-GGCTGGCT-3'
beginning at position -58 within the SSE element was also mutated to
include a ScaI site (5'-AGTACTAG-3') (Fig.
1). Approximately 260 bp of DNA
surrounding each of the mutations was sequenced on both strands, and it
was possible to verify that the mutation was present and to establish
that there were no other sequence changes.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 1.
-Globin promoter sequence.
The positions of the CACCC, two CCAAT, SSE, and TATA elements are
underlined. The Sp1 sites in the -50 and -140 regions are
not shown (51). The arrows indicate the positions of the
normal transcription start site (+1) and the two major start sites in
cells with the construct with the TATA box mutation, TATA ( 8), and
TATA ( 33).
|
|
Stable Transfection Assays and RNA and DNA Analyses--
HEL and
K562 cells were grown in RPMI media supplemented with 10% fetal calf
serum. For each of the six transfections per construct, 2 × 106 (HEL) or 1 × 106 (K562) cells were
electroporated with linearized plasmid DNA consisting of 35 µg of the
globin construct and 5 µg of an SV40 early promoter neomycin
resistance gene fusion construct. Two different plasmid preparations
per construct were transfected. Pools of transfected cells were
selected in 800 µg/ml and maintained in 600 µg/ml G418 sulfate
(Mediatech, Inc.). Pools of cells were used to analyze the results of
many different sites of insertion and thereby eliminate position
effects. Erythroid differentiation of the transfected HEL and K562
cells was induced by treatment with 25 µM hemin for 3 days. RNA was prepared from cells harvested after induction. mRNA
expression levels represent the average of at least two primer
extension assays quantitated using a Molecular Dynamics PhosphorImager.
The oligonucleotide primers used are
-globin
(5'-CAGGGCAGTAACGGCAGA-3'), which yields a 95-bp product for the
endogenous and a 99-bp product for the marked
-globin mRNA, and
-globin (5'-TGCCCCACAGGCTTGTGATA-3'), which yields a 105-bp product
for the endogenous and a 109-bp product for the marked
-globin
mRNA. The levels of transfected
- and
-globin mRNA are
expressed as a ratio of the endogenous
-globin mRNA as an
internal control. Sequencing experiments to determine the two major
alternative start sites for
-globin mRNA in cells with the
HS3HS2
TATA
constructs were performed using the fmol sequencing kit (Promega) and the above primer from the
-globin gene. The statistical analyses were performed using the nonparametric rank sum
test, and all findings were judged to be significant at an
-level of
0.05.
 |
RESULTS |
HS3 Exhibits Enhancing Activity but Has a Variable Effect on
Competitive Inhibition of
-Globin Expression--
Our previous
results indicate that HS2 can independently participate in competitive
interactions between the
- and
-globin genes in transgenic mice
and tissue culture cells (10, 13). This work tests the roles of HS3
alone and in combination with HS2 in these competitive interactions and
examines the possibility that these HS sites act in concert to enhance
- and
-globin expression in a developmental stage-specific manner.
To test the role of HS3 in the competitive inhibition of
-globin
expression, pools of HEL and of K562 cells were stably transfected with
each of two constructs containing HS3 and a marked
- or
- and
-globin gene (HS3
and HS3
, Fig.
2A). The transfected cells
were induced with hemin, and globin gene expression was measured by
primer extension assays using human
- and
-globin probes (Fig.
3). The amounts of mRNA from the
transfected genes (
mk and
mk) were normalized to that of the
endogenous
-globin mRNA. The results depicted in Table
I show that HS3 alone is an effective
enhancer and can drive significant expression of the transfected genes.
The transfected
-globin mRNA is expressed at means of 21 and
13% in HEL and K562 cells with the HS3
construct, respectively.
When comparing cells with the HS3
to those with the HS3
constructs, however, the presence of the
-globin gene in
cis does not significantly reduce
-globin expression in
HEL cells.
-Globin expression in HEL cells with and without the
-globin gene is comparable at an average of 7 and 4%, respectively
(Table I, Fig. 4). Although
-globin
expression is significantly lower on average in K562 cells with the
HS3
compared with the HS3
construct, there is variability
between pools of cells. Some pools of cells with HS3
have less
-globin mRNA than HS3
pools, but others do not, indicating
that HS3 alone does not consistently participate in competitive
inhibition in either cell type. The amount of transfected
-globin
mRNA was divided by the amount of transfected
-globin mRNA
for each pool of cells with the HS3
construct to calculate the
- to
-globin mRNA ratios (Table I). The
/
globin ratios
are particularly useful for comparisons between transfections, because
these values are not influenced by transfection efficiency. In HEL
cells, the mean
/
ratio is 26% even though the
-globin gene
is present in cis. The
-globin gene therefore does not
always compete strongly with the
-globin gene in the presence of HS3
alone. These data reveal that HS3 possesses enhancing activity, but
compared with HS2, it is less consistently able to participate in the
competitive interactions that normally suppress
-globin expression
in an early erythroid environment. The amount of the transfected
-globin gene expressed is generally less for all of the constructs
in K562 cells compared with HEL cells, perhaps because of the absence
of EKLF in K562 cells (33). For these and all of the transfections with
other constructs discussed below, the mRNA expression levels in the tables are not divided by the average number of copies of the genes per
cell, because in pools of cells with the same construct, a higher copy
number correlated directly with lower expression per gene copy (data
not shown).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
DNA constructs used to investigate
competitive inhibition in stable transfection assays in HEL and K562
cells. All of the constructs contain a 1.9-kb fragment containing
HS3 from the LCR and a 4.5-kb -globin gene. HS3HS2 , HS3HS2 ,
and the -globin promoter mutant constructs contain a 1.9-kb HS2
fragment. HS3 , HS3HS2 , and the -globin promoter mutant
constructs contain a 3.3-kb -globin gene. A, constructs
to determine the roles of HS3 and HS3HS2. B, constructs to
test the roles of the -globin TATA, CACCC, and SSE promoter
elements.
|
|

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 3.
Representative primer extension analysis of
RNA from HEL cells transfected with the HS3 ,
HS3 ,
HS3HS2 , and
HS3HS2 constructs. The
primer extension products are designated mk for the mRNA from
the transfected -globin gene; for the product of the endogenous
gene, and mk for the mRNA from the transfected -globin gene.
Each lane represents the RNA from a pool of cells from a
different transfection assay.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
- and -globin gene expression in HEL and K562 cells with HS3 and
HS3HS2 constructs
The mRNAs are designated mk for the transfected -globin gene,
mk for the transfected -globin gene, and for the product of
the endogenous gene. The percentages are the mean of at least two
primer extension assays. The means represent the average for all pools
of cells transfected with the same construct. Data for the HS3HS2
construct in HEL cells are pooled with those from Table II
(transfection numbers 4-9 corresponding to transfections 1-6 in Table
II and Fig. 5). The mk/ mk ratio is the amount of -globin
divided by -globin mRNA. The asterisks indicate that the mean
% mk and the mean mk/ mk ratios in each column are
significantly different from each other, that the HS3HS2 and
HS3HS2 transfections have a significantly different % mk, and
that the HS3 and HS3 transfections have a significantly
different % mk in K562 cells. The statistical analyses were
performed using the rank sum test, which is valid in cases where
the data do not fit a normal distribution.
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4.
The - to
-globin mRNA ratios for HEL and K562 cells
transfected with the HS3 and
HS3HS2 constructs. The -
to -globin mRNA ratio indicates the net effect of each LCR
configuration on the competition between the - and -globin genes.
The greater S.D. in the HS3 data series, depicted by the larger
relative size of the error bars, demonstrate the greater
variability with which HS3 participates in -globin gene
suppression.
|
|
HS2 and HS3 Act in Concert to Allow Stage-specific Expression of
the
- and
-Globin Genes--
To investigate the ability of HS3
and HS2 to act together in
-globin suppression in early development,
pools of HEL and of K562 cells were stably transfected with the
HS3HS2
and HS3HS2
constructs (Fig. 2A).
Representative primer extension assays for the HEL cells are shown in
Fig. 3. In contrast to the results with the HS3
and HS3
constructs,
-globin expression in both cell types is significantly
suppressed in the HS3HS2
compared with the HS3HS2
transfected
cells. HEL cells containing these constructs exhibit a 10-fold
suppression in
-globin expression (36.6% for HS3HS2
and 3.6%
for HS3HS2
), whereas K562 cells show a 4-fold suppression of
-globin expression (4.7 and 1.2%
-globin mRNA) (Table I).
The developmental specificity of this LCR configuration is also
demonstrated by the low
/
ratio in cells with HS3HS2
compared with those with HS3
(Table I, Fig. 4). The
/
ratios of 0.04 in HEL cells and 0.01 in K562 cells indicate that the
adult
-globin gene is consistently less able to compete with the
-globin gene when both HS3 and HS2 of the LCR are present than with
HS3 alone. These data demonstrate that HS2 and HS3 act together to
effectively provide developmental specificity.
HS2 and HS3 Together More Effectively Enhance
-Globin
Expression--
The effect of HS2 and HS3 on
-globin expression may
be elucidated by comparing the levels of
-globin mRNA in cells
with HS2
, HS3
, and HS3HS2
constructs. We have
previously shown that cells with HS2
express
-globin mRNA
at an average of 46 and 53% endogenous
-globin mRNA in HEL and
K562 cells, respectively (Ref. 13 and data not shown). HEL cells with
the HS3
construct express an average of 21%
-globin mRNA
compared with the endogenous
-globin mRNA, and those with
HS3HS2
express an average of 84%
-globin mRNA (Table I).
K562 cells with HS3
and HS3HS2
express 13 and 91%
-globin mRNA, respectively (Table
II). In both cell types,
-globin
expression with the HS3HS2
construct is significantly higher than
with HS3
. Therefore, HS2 and HS3 act in at least an additive
manner to effectively enhance
-globin gene expression.
View this table:
[in this window]
[in a new window]
|
Table II
- and -globin gene expression in HEL and K562 cells with
-globin promoter mutant constructs
The mRNAs are designated mk for the transfected -globin gene,
mk for the transfected -globin gene, and for the product of
the endogenous gene. The percentages are the mean of at least two
primer extension assays. The mean represents the average for all pools
of cells transfected with the same construct. The mk/ mk ratio is
the amount of -globin divided by -globin mRNA. The asterisks
indicate that the % or ratio differs significantly (using the
rank sum test) from that obtained with the wild-type construct,
HS3HS2 .
|
|
The Roles of the TATA, CACCC, and SSE Elements in the
-Globin
Promoter in Competitive Inhibition--
Our previous results are
consistent with the hypothesis that sequences 3' of base position
161
in the
-globin promoter must be present in cis to inhibit
embryonic expression of the
-globin gene (13). Mutations in elements
downstream of
161 have been tested in the context of a construct
containing HS3, HS2, the
1350
-globin gene and the
-globin
gene. HS2 and HS3 were included in the constructs to provide both
developmental specificity and strong enhancing activity.
Six transfections of the HS3HS2
, HS3HS2
TATA
,
HS3HS2
CACCC
, and HS3HS2
SSE
constructs depicted in Fig.
2 were performed in HEL and in K562 cells. Representative primer
extension analyses for the HEL cells are shown in Fig.
5. The most striking result from these
experiments is that the mutation of the TATA box in the
HS3HS2
TATA
construct leads to a 10-fold increase in the transfected
- to
-globin mRNA ratio compared with the
wild-type HS3HS2
in HEL cells (0.39 and 0.04, Table II, Fig.
6). This reflects both a 5-fold increase
in transfected
-globin mRNA and about a 2-fold decrease in
-globin mRNA on average (Table II). A similar though less
pronounced effect was evident in K562 cells; about a 2-fold increase in
transfected
-globin mRNA and a 3-fold decrease in
-globin
mRNA on average was observed with HS3HS2
TATA
compared with
HS3HS2
(Table II). These data suggest that factors that bind to
the TATA box are important for stabilizing interactions with HS3 and/or
HS2, which allow the
-globin gene to compete favorably with the
-globin gene in the embryonic/fetal environment.

View larger version (64K):
[in this window]
[in a new window]
|
Fig. 5.
Representative primer extension
analysis of RNA from HEL cells with the
HS3HS2 ,
HS3HS2 TATA ,
HS3HS2 CACCC , and
HS3HS2 SSE
constructs. The primer extension products are designated
mk for the mRNA from the transfected -globin gene; for
the product of the endogenous gene, and mk for the mRNA from the
transfected -globin gene. The arrows indicate major
alternative transcription start sites at base positions -8 and -33
for the -globin gene in the HS3HS2 TATA construct.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 6.
The - to
-globin mRNA ratios for HEL and K562 cells
transfected with the HS3HS2 ,
HS3HS2 TATA ,
HS3HS2 CACCC , and
HS3HS2 SSE
constructs. The - to -globin mRNA ratio indicates
the net effect of the -globin promoter mutations on the competition
between the - and -globin genes. The error bars
represent the S.D. in each data series.
|
|
It was somewhat surprising that the TATA mutation, which obliterates
the recognized consensus sequence, reduces
-globin gene expression
by only 2- to 3-fold compared with the HS3HS2
construct in HEL
and in K562 cells (see "Discussion"). The mutation leads to a great
reduction in transcripts beginning at the normal start site and to the
use of two major alternative transcription start sites in both HEL and
K562 cells, at positions
8 and
33 in the
-globin promoter (Figs.
1 and 5). The percentages calculated in Table II represent the sum of
all the mRNA products.
There is also a significant, greater than 2-fold increase in
-globin
transcription in the HEL cells with the HS3HS2
CACCC
construct
compared with those with the HS3HS2
construct (Table II). This is
accompanied by a small but significant decrease in
-globin gene
expression. In K562 cells, there is also a significant increase in
-globin mRNA and a significant decrease in
-globin gene
expression in cells with the HS3HS2
CACCC
construct compared with
cells with the HS3HS2
construct. The transfected
- to
-globin mRNA ratio was significantly increased by 2- or 3-fold in K562 or HEL cells with HS3HS2
CACCC
compared with those with HS3HS2
, representing a shift in the ability of the
- and
-globin genes to compete with each other. This indicates that the
CACCC element is also important in promoter competition. Again, it is somewhat surprising that the mutation in the CACCC element has only a
small effect on
-globin gene transcription, perhaps suggesting that
its role is redundant.
As can be seen in Table II, no significant change in the amount of
transfected
- or
-globin mRNA expressed resulted from the SSE
mutation in either HEL or K562 cells. The
- and
-globin genes in
cells with HS3HS2
SSE
were expressed at much the same level as in
cells with the HS3HS2
construct. The data suggest that the SSE
plays no role in competitive inhibition of
-globin gene expression
or at least that another element in the
1350
-globin gene can
compensate for the loss of the
50 SSE site.
 |
DISCUSSION |
We have demonstrated that HS2 and HS3 each contribute distinct
properties to the
-globin LCR. Although HS2 was previously shown to
participate in competitive interactions between the
- and
-globin
genes (10, 13), we find that HS3 does not consistently play a role in
these interactions in HEL and K562 cells. Both high level and
stage-specific expression of the
- and
-globin genes is observed
in cells with HS3HS2
constructs, because of the presence of both
HS3 and HS2. This work also demonstrates that multiple elements
downstream of -161 in the
-globin promoter, including the CACCC and
TATA but not the SSE, are required for competitive inhibition of
-globin gene expression.
Our evidence suggests that HS3 alone plays a less consistent role in
competitive inhibition than does HS2. This complements and expands upon
studies in which deletions of HS3 have no effect on the temporal order
of
-like globin gene expression (19). Although Fraser et
al. (18) observed developmental specificity of
- and
-globin
gene expression in two lines of transgenic mice with HS3 constructs,
this may not be a general phenomenon because the
-globin gene is
suppressed more in some pools of cells than in others in this study.
The greater ability of the HS3HS2 configuration, compared with HS2 (13)
or HS3 alone, to participate in competitive interactions between the
- and the
-globin genes strengthens the hypothesis that the HS
sites of the LCR act as a complex to activate transcription by
interacting with a single
-like globin gene at a time (7). The
results from our study using chromosomally integrated genes confirm
those of Jackson et al. (15), who found that HS2 and HS3
have additive enhancing activity in transient assays, and those of
Bresnick and Tze (16), who have shown that these sites are synergistic in long range enhancer activity in cells with chromosomally integrated genes (16).
The competition model states that the protein-protein interactions that
occur between factors binding to the LCR and to the
-globin promoter
preclude LCR interactions with the
-globin promoter (8, 9). The
finding that HS2 is more important than HS3 in competitive inhibition
directs further investigation toward the characterization of the roles
of proteins that bind to HS2. The data also suggest that proteins that
bind exclusively to HS2, and not to HS3, may be important for the
observed properties of HS2. The 215-bp core element of HS2 has a
cluster of binding sites for nuclear factors that appear to be
functionally required for enhancing activity (reviewed in Elnitski
et al. (34)) and may therefore take part in interactions
with the gene promoters. These proteins include NF-E2/AP-1, GATA-1,
YY1, USF (upstream stimulatory factor), and HS2NF5. The binding of
NF-E2/AP-1 provides much of the enhancing activity of HS2 (35, 36).
Mice lacking NF-E2 do not show impaired globin gene expression,
suggesting that related family members can compensate for its loss (37, 38). Binding sites for the erythroid factor GATA-1 occur in the HS2
core and in the
- and
-globin gene promoters. The levels of
GATA-1 vary during development, so it is possible that GATA-1 is
involved in developmental regulation (39). Furthermore, mutations in a
GATA-1 binding site reduce the enhancing activity of HS2 in transgenic
mice (40). The protein YY1, which demonstrates positive and negative
activities, interacts with the
-globin promoter (41) and with HS2.
USF is a broadly distributed E-box-binding protein that interacts with
HS2 and may facilitate preinitiation complex formation with other
transcription factors on globin promoters (42). HS2NF5, another
E-box-binding protein, provides some of the enhancing activity of HS2
(43, 44). USF and HS2NF5 have not been reported to bind to HS3, so they
are good candidates to facilitate the role of HS2 in competitive
inhibition. Of particular interest, HS2NF5 is the mammalian homolog of
a Notch-regulated transcription factor, and this further implicates its
potential for developmental control (44). None of these individual
sites is essential for the position-independent expression of the
-like globin genes, suggesting that chromatin-opening activity may
require the cooperation between these and other elements (45), some of
which may be upstream of the classical LCR (46). It is unlikely that a
single protein binding to or a single DNA element within HS2
independently directs the competitive inhibition of the
-globin gene.
It is not surprising that multiple
-globin promoter elements are
required to suppress inappropriately early
-globin gene expression,
because multiple promoter elements have previously been implicated in
the response to the LCR, including TATA, CCAAT, and CACCC (47). It is
possible that the CACCC element in the
-globin promoter plays a role
similar to the CACCC in the
-globin promoter. EKLF binds
preferentially to the
- rather than the
-globin CACCC (29). In
mice that have the human
-globin locus but lack EKLF, the expression
of the
-globin gene is reduced, and that of the
-globin gene is
increased (27, 28). Therefore, it is likely that the CACCC in the
-globin promoter is influential in switching to
-globin
expression, and we have now shown that the CACCC in the
-globin
promoter is also important for switching. Paradoxically, our work
indicates that a
-globin CACCC mutation can disallow
-globin
suppression without greatly reducing expression of the
-globin gene.
This is not the expected outcome from gene competition and suggests
that the model will need to be tested further. It is possible that the
CACCC and/or other as yet unidentified elements in the
-globin
promoter is responsible for initiating stage-specific interactions
between the
-globin gene and the LCR and that the TATA element is
important for stabilizing these interactions. The lack of a role for
the SSE in this system may reflect the fact that it has not previously
been tested using a chromosomally integrated construct or with an
intact globin gene rather than a reporter gene (31).
Curiously, the severe mutation we generated in the
-globin gene TATA
box reduced
-globin mRNA by only about 2- or 3-fold. However,
the two major transcription start sites utilized in cells with this
construct are shifted to positions further upstream than that for the
normal gene (-8 and -33, compared with +1, Fig. 1). A similar mutation
in the
-globin gene TATA box reduces transcription by 7-fold with no
aberrant start sites in stable transfection assays in MEL cells with
LCR constructs (48). Therefore, it seems likely that other elements in
the
-globin promoter can compensate in the absence of the ATAAAA at
base position -30. The chicken
-globin gene, for example, contains
a GATA-1 site at -30 rather than a TATA element. It can bind either
GATA-1 or TFIID and is necessary for transcriptional activation. (49) Potential GATA-1 binding sites (50) are located approximately 30 bases
upstream from each of the major aberrant start sites in cells with the
-globin promoter with the mutated TATA box (GAT at -38 and ATC at
-72, Fig. 1), and these may be comparable with that in the chicken
-globin gene. Alternatively, or perhaps additionally, the
-globin
promoter contains Sp1 binding sites in the -50 and -140 regions (51).
In TATA-less genes, Sp1 sites upstream from the start site are often
required for transcription (52). It is possible that the Sp1 sites in
the
-globin promoter are utilized in a similar manner in the absence
of the TATA box. Our data predict that human mutations in the
-globin TATA box may not be seriously deleterious.
In previous work, it was suggested that any transcribed gene
intervening between the
-globin gene and the LCR might suppress
-globin expression. The
-globin gene was suppressed in embryonic transgenic mice containing either an
-globin (53) or a thymidine kinase-chloramphenicol acetyl transferase construct (26). However, Sabatino et al. (54) demonstrated that specific sequences in the
-globin promoter are required for competitive inhibition, because a
-globin gene with a
-spectrin promoter does not
suppress
-globin expression in embryonic transgenic mice, even
though it is transcribed (54). This evidence supports the hypothesis that competitive inhibition of the
-globin gene is mediated by specific
-globin promoter-LCR interactions, rather than by
transcriptional interference by a transcribed gene situated between the
LCR and the
-globin gene (55). It is possible that the thymidine
kinase-chloramphenicol acetyl transferase construct and
-globin
constructs, which can compete, contain sequences that allow these genes
to interact with and sequester the LCR. It was shown that the
expression of the
-spectrin/
-globin fusion construct was not
influenced by the LCR, suggesting that it does not contain such
sequences (54). We have shown that the TATA and CACCC elements are
necessary for efficient
-globin competition, but we suggest that
they are probably not sufficient. Although the
-globin,
-globin,
thymidine kinase, and
-spectrin promoters all contain TATA and
CACCC/EKLF elements, the
-spectrin promoter cannot compete (56-58).
Therefore, it is likely that the precise positions of these elements,
and/or that other elements, are also important for gene competition.
By identifying the specific sequences in the
-globin promoter and
LCR that are required for
-globin gene suppression in early
erythroid development, candidate proteins that are involved will be
implicated for future investigation. This will further delineate the
molecular mechanisms of developmental regulation of multiple genes by a
distant enhancer.