(Received for publication, September 14, 1994; and in revised form, November 4, 1994)
From the
Erythroid Kruppel-like factor (EKLF) is an erythroid-specific
transcription factor that contains zinc finger domains similar to the
Kruppel protein of Drosophila melanogaster. Previous studies
demonstrated that EKLF binds to the CACCC box in the human -globin
gene promoter and activates transcription. CACCC box mutations that
cause severe
-thalassemias in humans inhibit EKLF binding. Results
described in this paper suggest that EKLF functions predominately in
adult erythroid tissue. The EKLF gene is expressed at a 3-fold higher
level in adult erythroid tissue than in fetal erythroid tissue, and the
EKLF protein binds to the human
-globin promoter 8-fold more
efficiently than to the human
-globin promoter. Co-transfection
experiments in the human fetal-like erythroleukemia cell line K562
demonstrate that over-expression of EKLF activates a
-globin
reporter construct 1000-fold; a linked
-globin reporter is
activated only 3-fold. Mutation of the
-globin CACCC box severely
inhibits activation. These results demonstrate that EKLF is a
developmental stage-enriched protein that preferentially activates
human
-globin gene expression. The data strongly suggest that EKLF
is an important factor involved in human
- to
-globin gene
switching.
All vertebrate animals switch hemoglobins during development. In
humans, the first site of erythropoiesis is the yolk sac blood islets.
Erythroid cells in the yolk sac are formed from embryonic mesoderm at
approximately 3 weeks of gestation. The first hemoglobins produced by
these cells are tetramers composed of 2 -globin or 2
-globin
polypeptides and 2
-globin polypeptides. The hemoglobins are
designated Gower I (
) and Gower II
(
).
-Globin gene expression
gradually decreases and
-globin expression gradually increases
over the next few weeks until Gower II
(
) is the predominate hemoglobin. At
approximately 5 weeks of development, hematopoietic stem cells from the
yolk sac migrate to the fetal liver and initiate hematopoiesis in this
organ. Fetal liver then becomes the major site of erythropoiesis, and
there is a concomitant switch in hemoglobin production;
-Globin
gene expression decreases, and the fetal
-globin gene is
activated. The major hemoglobin produced at this stage of development
is, therefore, fetal hemoglobin (
).
Finally, hematopoietic stem cells from the fetal liver migrate to the
bone marrow. This organ then becomes the major site of erythropoiesis,
and there is a final switch in hemoglobin synthesis. Expression of the
-globin gene steadily decreases, and expression of the
- and
-globin genes gradually increase until approximately 98% of total
hemoglobin is hemoglobin A (
) and
approximately 1% is hemoglobin A
(
); the
-globin promoter
is enfeebled and, therefore, is transcribed poorly. The
-globin
gene continues to be transcribed in a minor population of erythroid
cells that develop in the adult bone marrow; these cells are designated
F cells, and the HbF in these cells accounts for approximately 1% of
total hemoglobin in adult blood(1) .
The molecular
mechanisms that direct hemoglobin switching during development are
almost completely unknown. In humans, a powerful regulatory sequence
designated the locus control region (LCR) ()is located far
upstream of the
-globin locus on chromosome 11 (for review, see (2, 3, 4, 5) )). The
-globin
LCR has two important functions. First, these sequences open a
chromosomal domain that extends over 200 kb. This chromatin
decondensation renders individual genes in the locus more accessible to trans-acting factors that control temporal specific
expression. Secondly, the LCR acts as a master enhancer; individual
globin gene family members compete for interactions with the LCR to
determine which genes are expressed at specific developmental stages.
Positive and negative regulatory factors that bind to specific globin
gene promoters and proximal enhancers or silencers provide individual
genes with a competitive advantage or disadvantage for interaction with
the LCR. Once interactions between a particular globin gene(s) and the
LCR is established, the complex is relatively stable and commits this
chromosomal allele(s) to express
, G
, and A
or
-
and
-globin throughout the lifetime of the cell.
Although
developmental stage-specific factors have been proposed to regulate
globin gene expression during development, no proteins that control
human - to
-globin gene switching have been identified. In
this paper, we have examined the role of erythroid Kruppel-like factor
(EKLF) in globin gene switching. EKLF is an erythroid cell-specific
transcriptional activator that contains three zinc fingers homologous
to the Kruppel family of transcription factors(6) . As shown by
crystallographic and ``finger-swapping'' experiments with
other members of this family, each finger contacts 3 base pairs, such
that the binding site for the EKLF protein was predicted to be
3`-GGNGNGGGN-5`. Based on this information, it was demonstrated that
EKLF binds to and mediates its transcriptional activation via the human
and murine adult
-globin CAC site (5`-CCACACCCT-3`)(6) , a
site known to be critical for
-globin
expression(7, 8, 9, 10) .
Methylation interference studies indicated that EKLF forms close
contacts to each of the guanine residues within this extended 9-base
pair site, such that point mutation of some of these residues,
including those that give rise to
-thalassemia, drastically
disrupt binding(11) . These results suggest that EKLF may be
intimately involved in the regulation of globin expression through its
interaction with the CACCC element.
However, inspection of
CAC-related sequences in other murine and human -globin promoters
in the context of the complete 9-base pair EKLF-binding element reveals
that these sites do not form a homogeneous group. Of particular
interest for the present study is the sequence 5`-CTCCACCCA-3` in the
human fetal A
- and G
-globin promoter (and in the murine
embryonic y-globin promoter). This sequence contains a mismatch to the
predicted EKLF binding site, i.e. binding by the EKLF
amino-terminal finger to 5`-CCN-3`. Our previous studies have
emphasized the importance of interactions by specific nucleotides in
the complete
-CAC site with critical EKLF amino acid
residues(6, 11) . A decrease in EKLF binding affinity
to the variant
-CAC site could play a major role in determining
the relative levels of
- and
-globin transcription. We have
therefore directly tested the ability of EKLF to bind to the
-globin CAC site in vitro and have examined the role of
EKLF in human
- to
-globin gene switching in vivo.
HS 2-/luciferase-
/CAT was
constructed in several steps.
/CAT was made by inserting the
blunt-ended
-globin promoter fragment described above into the
plasmid pCAT-Basic (Promega), which had been cut with XbaI and
blunted with Klenow polymerase. The following three fragments were then
ligated to make HS 2-
/luciferase-
/CAT: a 4.5-kb KpnI-SalI fragment from HS 2-
/luciferase
containing HS 2, the
-globin promoter, the luciferase gene, and
the SV40 splice and polyadenylation signals; a 2.0-kb SalI-BamHI
/CAT fragment containing the
-promoter, CAT gene, and splice and poly(A) signals; and a 2.9-kb BamHI-KpnI fragment from pGL2-Basic (Promega)
containing prokaryotic vector sequences.
To construct HS 2
(CACCC)-
/luciferase-
/CAT, a mutant HS 2
fragment containing the scrambled CACCC motif was derived from a
previously described plasmid (13) (plasmid 5`-HS 2 (K-P)
8689-98s)). The 1.4-kb KpnI-StuI HS 2
fragment from this mutant plasmid was used to replace the corresponding
wild type region in HS 2-
/luciferase-
/CAT.
To make HS
2-/luciferase-
(-87)/CAT, a
-promoter containing
the -87 G to C
-thalassemia mutation was constructed using
the megaprimer mutagenesis
method(14, 15, 16) . The outside primers
overlapped the SnaBI site at -265 and the NcoI
site at +48 of the human
-promoter. The 3`-primer changed the NcoI site to a SnaBI site (underlined in the sequence
below) so that a blunt end promoter fragment could be easily prepared.
The template was a linearized HinfI subclone of the human
-promoter in pUC 19. Primers used to amplify the mutant promoter
were 1) the upstream pUC reverse primer from New England Biolabs, 2)
the mutagenic oligonucleotide
5`-CCTGGGAGTAGATTGGCCAACCCTAGCGTGTGGCTCCACAGGGTGAGGTCTAAGT-3` (the
mutated base is underlined), and 3) the downstream oligonucleotide
5`-AGGTGCACCTACGTATCGGTTTGAGGTTGCTAGTG-3` (the underlined bases
represent a SnaBI site). The resulting SnaBI fragment
containing the
(-87) mutation was used to make HS
2-
/luciferase-
(-87)/CAT as described for the wild type
plasmid. All promoter sequences were verified by dideoxy sequencing (17) using the Sequenase kit (U. S. Biochemicals Corp.).
Fig. 1A illustrates a competitive gel shift
experiment designed to measure the relative binding efficiency of EKLF
to the CACCC boxes in the human - and
-globin gene promoters.
A double-stranded oligonucleotide containing the
-globin CACCC box
was end-labeled and incubated with purified EKLF (11) in the
presence of increasing amounts of unlabeled
- and
-globin CAC
box oligonucleotides. The results were quantitated and graphed as
illustrated in Fig. 1B. Under the conditions of the
assay, an 11-fold excess of
-globin CAC site was required to
inhibit the EKLF-CAC shift by 50%; however, a 90-fold excess of the
-globin CAC site was required for 50% inhibition. These results
demonstrate that EKLF binds approximately 8-fold more efficiently to
the
-globin CACCC box than to the
-globin CACCC box; this is
consistent with the very weak EKLF/
-CAC gel shift that is observed
relative to that seen with EKLF/
-CAC (Fig. 1C).
The higher binding affinity of EKLF to adult versus fetal
globin gene promoters suggests that EKLF may be involved in
- to
-globin gene switching.
Figure 1:
Competitive gel retardation
analysis of EKLF binding to variant CAC site-containing
oligonucleotides in vitro. Gel shift assays used radiolabeled
adult -globin oligonucleotides and the indicated non-radioactive
competitor oligonucleotides at 0-, 20-, 50-, 100-, 200-, and 400-fold
molar excess (lanes2-7, respectively). Lane1 contained no protein added to the incubation.
Data for two preparations of purified EKLF are shown to demonstrate the
reproducibility of the assay. The autoradiograph of the gel resulting
from all the assays is shown in A, and its quantitation is
shown in B. The amount of shift seen without any competitor is
defined as 100%. The point at which each of these curves crosses the
``50% signal remaining'' line was used as the basis for
estimating the competitive ability of each oligonucleotide for binding
to EKLF relative to adult
-globin CAC. C, direct binding
analyses of EKLF and radiolabeled
- or
-CAC site-containing
oligonucleotides in vitro are shown. The specific activities
of these probes were equivalent, and equal counts/min were loaded in
each lane.
The level of EKLF expression in fetal
and adult erythroid tissue was also examined. Fig. 2illustrates
a Northern blot of mouse yolk sac, fetal liver, and reticulocyte RNA
probed with the murine EKLF cDNA clone. The filter was subsequently
stripped and reprobed with a mouse -globin clone as a control.
Bands were quantitated on a phosphorimager, and EKLF expression was
normalized to
-globin expression. The results demonstrate that
EKLF expression in mouse fetal liver, which is an adult erythroid
tissue, is 3-fold higher than expression in mouse yolk sac. The switch
from embryonic/fetal globin to adult globin gene expression in the
mouse occurs when the site of erythropoiesis shifts from yolk sac to
fetal liver at approximately 14 days of development; adult globin
expression is then maintained when bone marrow becomes the major site
of erythropoiesis at birth. The higher levels of EKLF mRNA in adult
compared with fetal tissue also suggest that EKLF may be involved in
- to
-globin switching.
Figure 2:
Northern blot analysis of EKLF and mouse
-globin expression. Lanes1-6 were loaded
with 2 µg of total RNA from the indicated cell lines and tissues. Lane1, human erythroleukemia cells (K562); lane2, mouse erythroleukemia cells uninduced (MEL-U); lane3, mouse erythroleukemia cells
induced with 1.5% dimethyl sulfoxide for 3 days (MEL-I); lane4, yolk sacs dissected from 10.5-day-old mouse
embryos (10.5 d YS); lane5, fetal liver
dissected from 16-day-old mouse fetuses (16 d FL); lane6, adult blood from phenylhydrazine-treated mice (Ad.
Blood). Phosphorimager quantitation of bands (Molecular Dynamics
Phosphorimager) shows that the level of EKLF message/
-globin
message is 3-fold higher in 16 d FL than in 10.5 d YS. A faint band of
EKLF and
-globin mRNA is observed in the K562 lane after longer
exposure (data not shown).
To determine the functional
consequences of differential EKLF binding to human - and
-globin gene promoters, co-transfection experiments in K562 cells
were performed. These cells normally synthesize little EKLF (Fig. 2), and no
-globin mRNA can be detected (data not
shown).
-Globin/luciferase (
/Luc) and
-globin/CAT
(
/CAT) reporter genes were inserted downstream of the LCR HS 2 (18) (Fig. 3A), and these constructs were
co-transfected with an EKLF expression vector into K562 cells. Fig. 3B demonstrates that EKLF stimulates
/Luc
expression only 3-fold; however,
/CAT expression is stimulated
30-fold (Fig. 3B). These results demonstrate that EKLF
preferentially activates
-globin gene expression, and the data
suggest that preferential binding of EKLF to the
-globin gene
CACCC box is at least partially responsible for this effect.
Figure 3:
EKLF transactivation analysis of
individual HS 2 /Luc and HS 2
/CAT reporter constructs. A, reporter constructs used to transfect K562 cells. HS
2-
/luciferase has been previously described(12) . This
plasmid contains the 1.5-kb KpnI-BglII HS 2 fragment
cloned upstream of a -299 to +36 human
-globin promoter
driving the luciferase gene. HS 2-
/CAT contains the identical
1.5-kb KpnI-BglII HS 2 fragment cloned upstream of a
-265 to +48 human
-globin promoter driving the
chloramphenicol transacetylase gene. The transactivator plasmid
SV40-EKLF has been described (pSG5-EKLF(6) ). B,
transactivation results. Reporter plasmids were transfected into K562
cells with or without SV40-EKLF and an internal control plasmid
pTK-
-galactosidase (Clontech) as described by Caterina et
al.(12) . Luciferase and CAT activities were normalized to
-galactosidase levels. EKLF enhanced HS 2-
/CAT 30-fold and HS
2-
/Luc only 3-fold. An HS 2-
/Luc construct was also tested in
K562 cells so that a direct comparison of
- and
-globin
promoter activities could be made (data not shown). Without exogenous
EKLF, the
-globin promoter was 27-fold less active than the
-globin promoter.
Competition models for human - to
-globin gene switching
predict that there are adult-specific or adult-enriched positive
regulatory factors that bind to the
-globin gene and give this
gene a preferential advantage to form interactions with the LCR. The
LCR then enhances high level expression of the
-globin gene
specifically in adult erythroid cells. To test the effect of EKLF on
linked
- and
-globin genes, we co-transfected K562 cells with
an EKLF expression vector and a construct containing
/Luc and
/CAT reporters inserted downstream of LCR HS 2 (HS 2
/Luc-
/CAT) Fig. 4A). Again, EKLF expression
stimulated the
-globin promoter only 3-fold; however, the
-globin promoter was enhanced 1,000-fold (Fig. 4B). These data suggest that EKLF binding to the
-globin CACCC box may play a critical role in human
- to
-globin gene switching. When the HS 2
/Luc-
/CAT
construct was transfected into Hela cells with and without the EKLF
expression vector, no enhancement of
- or
-reporter
expression by EKLF was observed (data not shown).
Figure 4:
EKLF transactivation analysis of linked
/Luc and
/CAT reporter genes. Transfections were performed as
in Fig. 3, except that the amount of reporter plasmid was
adjusted to maintain a 10-fold molar excess of transactivator plasmid.
EKLF stimulated
/CAT activity 1050-fold and
/Luc activity
only 3-fold. A direct comparison of
/CAT activities from HS 2
/Luc-
/CAT transfections and HS 2-
/CAT transfections were
also made (data not shown). In the absence of exogenous EKLF,
/CAT
activity from the HS 2
/Luc-
/CAT reporter was 120-fold lower
than
/CAT activity from the HS 2
/CAT
reporter.
As described
above, a mutation at -87 (CACCC to CACGC) in the -globin
promoter inhibits
-globin gene expression and causes
-thalassemia in humans(19, 20) . This -87
mutation was introduced into the
-globin gene promoter in the
linked
/Luc-
/CAT reporter construct, and the plasmid was
co-transfected with the EKLF expression vector into K562 cells. The
data in Fig. 5demonstrate that the -87 mutation strongly
inhibits EKLF activation of the
-globin gene (1000 to 4-fold
activation). Mutation of the phylogenetically conserved CACCC box (21) located approximately 15 base pairs downstream of the
Ap1-like sites in HS 2 also decreases
-globin gene activation
(1000 to 730-fold activation).
Figure 5:
EKLF transactivation analysis of the HS 2
/Luc-
/CAT reporter construct containing CACCC box mutations.
The phylogenetically conserved HS 2 CACCC box was scrambled in HS 2
(CACCC
)-
/Luc-
/CAT as described under
``Materials and Methods.'' HS 2-
/Luc-
(-87)/CAT
contains a single C to G point mutation at -87 of the
-promoter. This mutation is known to cause
-thalassemia in
humans and to inhibit EKLF binding in
vitro(11) .
Models of human globin gene switching postulate that
developmental stage-specific transcription factors bind to promoters
and proximal enhancers or silencers and influence the interaction of
-,
-, and
-globin genes with the powerful LCR. Although
these stage-specific proteins have been postulated for many years, no
positive or negative regulatory factors that direct
- to
-globin gene switching have been identified. The data described
above strongly suggest that EKLF is a developmental stage-enriched
factor that is involved in the switch from human
-globin to
-globin gene expression. The binding affinity of EKLF is 8-fold
higher for the
-globin promoter than for the
-globin promoter (Fig. 1), and the EKLF gene is expressed at a 3-fold higher
level in adult erythroid tissue than in fetal erythroid tissue (Fig. 2). Although we have not yet quantitated EKLF protein in
these cells, Northern blot data (Fig. 2) suggest that EKLF
levels in adult erythroid tissue are significantly higher than in fetal
erythroid tissue. To determine the functional consequences of
differential EKLF concentration and binding affinity, we co-transfected
an EKLF expression vector with HS 2
/Luc and HS 2
-/CAT
reporter constructs into K562 cells. These fetal-like erythroleukemia
cells express
- but not
-globin genes and normally synthesize
little EKLF (Fig. 2). After transfection,
-Luc was
activated only 3-fold, but
/CAT was activated 30-fold (Fig. 3). When
/Luc and
/CAT were linked in the same
construct (HS 2
/Luc-
/CAT) and co-transfected with the EKLF
expression vector into K562 cells, the
-globin promoter was
activated 1000-fold; the
-globin promoter was activated only
3-fold (Fig. 4). Mutation of the
-globin CACCC box at
-87 (CACCC to CACGC) strongly inhibited EKLF activation (1000 to
4-fold activation Fig. 5). These results suggest that EKLF is an
important factor in human
- to
-globin gene switching and
that the CACCC boxes are critical elements in this switch. Mutation of
the phylogenetically conserved CACCC site in HS 2 modestly inhibits
EKLF activation in transient assays (Fig. 5). Analysis of this
same mutation in transgenic mice shows a similar reduction in
activity(13) . Reddy et al.(22) recently
showed that the HS 2 CACCC site is footprinted in vivo in
adult human erythroblast but not in the fetal environment of K562
cells. These results, together with the results presented in this
paper, suggest that the HS 2 CAC site may also contribute to
-globin gene activation.
The lower binding affinity of EKLF for
the -globin CAC site is not entirely unexpected. Methylation
interference demonstrates that EKLF interacts with all the guanine
residues on the G-rich strand of the
-globin CAC site, including
the sequence 3`-GGN-5`, which is the putative first finger target
site(11) . These studies showed that changes of single guanine
residues had a dramatic effect on EKLF binding affinity to those
variant sites. In the
-CAC site, the first finger target site
would be 3`-GAG-5`, yielding a loss of an important guanine residue.
The lower affinity for this site indicates that binding of the
amino-terminal EKLF zinc finger is an important contributor to the
overall affinity of EKLF-CAC site interaction. As a result, efficient
EKLF binding to the CTCCACCCA site present in the
-globin promoter
is very low and may be heavily dependent on the effective EKLF protein
concentration or on the presence of a cofactor. Alternatively, Ikuta
and Kan (23) have demonstrated an in vivo footprint on
the
-CAC box but not on the
-CAC box in K562; therefore,
transcription of the
-globin gene may require another CACCC
element-binding factor that is primarily active in fetal, rather than
adult, erythroid cells.
Competition models of globin gene switching
predict that enhanced interaction of one globin gene with the LCR
necessarily decreases the interaction of another gene with the LCR.
This mechanism accounts for the precise developmental specificity of
globin gene expression. However, in the experiments described above,
both - and
-globin genes were stimulated. EKLF expression
enhanced
/CAT activity 1000-fold, and
/Luc activity did not
decrease but increased 3-fold. Stimulation of both genes in this
instance may occur because all of the regulatory factors required for
expression of
- and
-globin genes are present at the same
time. K562 cells normally contain the factors necessary for
-globin expression, and ectopic expression of EKLF apparently
provides an additional factor necessary for
-globin gene
activation. Expression of both genes in the same cell would be the
predicted result if the equilibrium constants for LCR-
and
LCR-
interactions are equivalent when both fetal and adult
regulatory factors are present.
If EKLF was the only positive factor
necessary for - to
-globin gene switching, one would predict
that overexpression of this factor in fetal erythroid cells would
activate the endogenous
-globin gene. However, we stably
transformed K562 cells with the EKLF expression vector, and no
endogenous
-globin mRNA was detected. This result suggests that
additional factors are required to activate a chromosomal copy of the
adult gene. Although the entire
-globin locus appears to be in an
``open'' or DNase I-sensitive domain in erythroid cells,
local changes in chromatin structure around individual genes may play a
role in switching. Perhaps additional temporal specific factors are
required to reposition nucleosomes so that the CACCC boxes are more or
less accessible to EKLF(24, 25, 26) .
Jane et al.(27) recently defined a stage-selector element
in the -globin promoter that appears to be important in
-globin gene activation. This sequence is located between
-54 and -35, and insertion of the stage-selector element in
a
-globin gene construct results in a 10-fold increase in
-globin gene expression in K562 cells. A fetal-specific protein
complex designated SSP (stage-selector protein) binds to the sequence (28) and is most likely involved in
-globin gene
activation in fetal development. Therefore, this protein and EKLF may
be critical fetus- and adult-specific proteins that are responsible for
human
- to
-globin and
- to
-globin gene switching
during development.
The results described above strongly suggest that EKLF is an important factor in temporal control. Targeted mutation of the EKLF gene in embryonic stem cells should provide additional information on the role of EKLF in hemoglobin switching. Based on the data in this paper, the phenotype of mice that are homozygous for a mutation in EKLF can be predicted. These mice should survive through early development but then die between 12 and 14 days of gestation when the switch from fetal to adult globin gene expression occurs.