Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2259-2263
Erythroid Krüppel-Like Factor Is Essential for 
-Globin Gene
Expression Even in Absence of Gene Competition, But Is Not
Sufficient to Induce the Switch From 
-Globin to -Globin Gene
Expression
By
Louis-Georges Guy,
Qi Mei,
Andrew C. Perkins,
Stuart H. Orkin, and
Lee Wall
From the Centre de recherche du Centre hospitalier de
l'Université de Montréal; the Institut du cancer de
Montréal, Montreal, Quebec, Canada; the Division of
Hematology/Oncology, Children's Hospital; the Dana Farber Cancer
Institute; Howard Hughes Medical Institute, Boston, MA; the Department
of Medicine, Université de Montréal, Montreal, Quebec,
Canada; and the Department of Physiology, Monash University, Australia.
 |
ABSTRACT |
Different genes in the 
-like globin locus are expressed at
specific times during development. This is controlled, in part, by
competition between the genes for activation by the locus control region. In mice, gene inactivation of the erythroid Krüppel-like factor (EKLF) transcription factor results in a lethal anemia due to a
specific and substantial decrease in expression of the fetal/adult-stage-specific -globin gene. In transgenic mice
carrying the complete human -globin locus, EKLF ablation not only
impairs human -globin-gene expression but also results in increased
expression of the human 
-globin genes during the fetal/adult stages.
Hence, it may appear that EKLF is a determining factor for the
developmental switch from -globin to -globin transcription.
However, we show here that the function of EKLF for -globin-gene
expression is necessary even in absence of gene competition. Moreover,
EKLF is not developmental specific and is present and functional before the switch from -globin to -globin-gene expression occurs. Thus, EKLF is not the primary factor that controls the switch. We suggest that autonomous repression of -globin transcription that occurs during late fetal development is likely to be the initiating event that
induces the switch.
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INTRODUCTION |
IN MAMMALS, EXPRESSION of globin genes is
erythroid specific and is developmentally regulated.1 The
human -globin locus contains five active genes: 
, which is
transcribed in the embryonic yolk sac; G and A, which are
expressed in the fetal liver; and 
(a minor contributor) and ,
which are transcribed in the bone marrow throughout adult life. These
five genes are arranged in their temporal order of expression
(5
--G-A---3; Fig 1). There is only one
well-defined switch in globin gene expression from the mouse -like
globin locus. Mice express y, h0, and h1 globins
during embryonic life and in early fetal development. Thereafter,
expression switches to the -minor and -major globins genes, and
these two genes continue to be transcribed into adult life.
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| Fig 1.
Structure of the human -like globin locus and the
-globin gene minimal promoter. The figure also shows EKLF that
interacts with the CACC motif element of the promoter.
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For high-level expression, the -like globin genes are dependent on a
locus control region (LCR). The LCR in the -globin locus is
signified by four DNase I hypersensitive site regions situated upstream
of the genes (Fig 1). This DNA element has very strong
erythroid-specific enhancer activity and is characterized as an LCR by
its ability to confer copy-number-dependent,
integration-site-independent, endogenous levels of expression onto
globin transgenes.2-4
In mice, transcription of a human -globin transgene present alone in
cis with the LCR is downregulated, but only partially, during
fetal development.5-9 The human -globin gene is
expressed at equivalent levels at all developmental stages in
transgenic mice when it is present alone with the LCR.6,7
On the other hand, if -globin and -globin genes are linked
together with the LCR (LCR--), both genes are appropriately
regulated during development; the -globin gene is not expressed in
the embryo and then is switched on near 11.5 days in fetal life,
whereas the -globin gene is expressed in the embryo and then is
markedly downregulated during fetal life when the -globin gene is
upregulated.6,7,10 These observations, in conjunction with
several other experiments,11-15 have suggested a
competitive model to describe the developmental regulation of globin
genes. It has been postulated that a globin gene must interact directly
with the LCR through a looping mechanism for it to be activated and
that the LCR can only interact with one gene at a time. Thus, the
globin genes compete with each other to contact the
LCR.6,7,10 At each developmental stage, the array of
proteins that interact with the LCR and/or the globin genes
favors interaction with a particular gene. As switching takes place,
modifications in the transcription factor environment alter the
relative affinity between the LCR and the genes such that the
interaction switches from one gene to the next. Therefore, competition
between the globin genes partly dictates their developmental pattern of
expression.6,7,9 Identifying the transcription factors
involved in LCR-gene interactions and/or the switching process
would be a major step toward elucidating the mechanism regulating
globin gene developmental specificity and the LCR mode of action.
The erythroid Krüppel-like factor (EKLF) plays an important role
in transcription of the mouse fetal/adult- and human
adult-stage-specific -globin genes. Upon binding to the CACC box
element in the promoters of these globin genes (Fig 1), EKLF can act as
a transcriptional activator.16-20 Mice in which the EKLF
gene is knocked out suffer from a severe anemia and die at
approximately 16 days postcoitus.21,22 The anemia is caused
by a major (approximately 20-fold) downregulation in expression of the
mouse -major gene. Other erythroid-specific genes, including other
globin genes, are expressed at normal levels, and erythropoiesis is
apparently otherwise unaffected. Therefore, the requirement for EKLF
function appears to be very specific to the fetal/adult-specific
-major globin gene in mice. Similarly, in transgenic mice carrying
the complete human -like locus, expression of the human -globin
gene is severely hampered in an EKLF
/
background.15,23 However, in the absence of EKLF the human -globin transgenes are actually expressed at higher-than-normal levels during fetal life, up to the time of embryonic lethality. This
result indicates that the switch in LCR interaction from the -globin
to the -globin gene that is expected to occur in early fetal
development in mice may not take place in the absence of EKLF.
A model to account for the above results would suggest that EKLF is
directly implicated in the interaction of the -globin gene with the
LCR and that EKLF activity is developmental specific. EKLF does
interact with the erythroid-specific protein GATA-1,24,25 for which there exist many DNA-binding sites in the
LCR.26-29 Thus, there is a potential for EKLF to be
involved in LCR-gene interactions. Whether or not the function of EKLF
is developmentally regulated has not been tested directly, although it
is known that the mRNA is present at early stages.20,30 On
the other hand, the fact that the -globin gene is expressed at all
stages of development when it is alone with the LCR (see above)
questions the requirement for a developmental-specific modifier acting
on the -globin gene and involved in the switching process. EKLF
function might, therefore, only be necessary for competition against
other globin genes. To address these questions, we have looked at EKLF
function in the absence of gene competition and during development.
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MATERIALS AND METHODS |
Transgenic mice.
EKLF+/ mice and µD14 mice, which have been described
previously,22,31 were maintained on a C57B16 × 129 and
C57B16 × CBA background, respectively. Fetuses were dissected out at
the indicated times following the observation of the spermatic plug.
DNA analyses.
Fetal DNA was digested with HindIII, separated on agarose gels,
and blotted onto nylon membranes. The membranes were hybridized to an
Nco I-Sma I 550-bp fragment from the 5 region of EKLF
cDNA that distinguishes between the wild-type and the targeted EKLF allele. The membranes were rehybridized with a 750-bp HindIII fragment from HSS2 of the LCR that detects this same 750-bp fragment from the µD transgene.
RNA analyses.
RNA was isolated using Trizol reagent (GIBCO-BRL, Gaithersburg,
MD) as described by the manufacturer. RNase protection
assays were conducted as described32 with antisense probes
that span the 5 region of the mRNA.
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RESULTS |
The -globin gene requires EKLF for expression even when
it is juxtaposed to the LCR and no gene competition is present.
In the complete -like loci, the LCR is about 60 kb away from the
-globin gene (Fig 1). There are several genes that compete with the
-globin gene for interaction with the LCR, and this competition
plays a role in the developmental regulation of these genes.6,7,10 Here, we aimed to test the function of EKLF in
-globin-gene expression in the absence of gene competition.
We studied a transgenic mouse line carrying the human microlocus
construct33,34 in which the four hypersensitive site
regions of the LCR are directly linked in cis to the complete
human -globin gene. In this construct, the LCR is within 4 kb of the
TATA box of the -globin-gene promoter, as measured from the middle
of DNase hypersensitive sites 2 and 3 of the LCR. The expression of
this transgene is erythroid specific and position
independent.33 We used the µD14 transgenic line, which
carries a single copy of the transgene and expresses human -globin
at 43% the level of the endogenous mouse -major gene.31
µD14 transgenic mice were bred to mice heterozygous for the EKLF
inactivated allele. F1 animals that were transgenic and
EKLF+/ were bred again to EKLF heterozygous mice to
obtain µD14 fetuses null for the EKLF gene. The genotype of the
animals was determined by Southern blot using a probe that
distinguishes the EKLF wild-type allele from the knockout allele and a
probe that is specific for the human LCR region of the transgene (Fig
2). Human
-globin-transgene expression was measured relative to mouse
-major mRNA via RNase protection (Fig
3). Comparison of human -globin mRNA
levels in fetal livers from EKLF knockout, heterozygous, and wild-type
animals showed that expression of the transgene is as dependent on EKLF as the endogenous mouse -major gene. Both genes gave 15- to 20-fold lower expression in the absence of EKLF. In comparison, GATA-1 expression was not affected by EKLF inactivation (Fig 3). Thus, although the -globin promoter is juxtaposed to the LCR and no gene
competition exists in the µD transgene, -globin expression is
still highly dependent on EKLF.
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| Fig 2.
Genotype determination. Fetus DNA digested with
HindIII was analyzed by Southern blot with an EKLF probe that
distinguishes between the knockout allele (10-kb fragment
detected) and the wild-type allele (6-kb fragment detected). The same
blot was reprobed with human LCR sequences that hybridize to a 0.75-kb
HindIII fragment from the transgene. The EKLF genotype and the
presence (tg) or absence (nt) of the transgene is indicated at the top
of each lane.
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| Fig 3.
EKLF is required for -globin-transgene expression in
the fetal-adult stage. Fourteen-and-a-half-day fetal liver RNA was
isolated from µD14 transgenic (tg) animals or nontransgenic (nt)
litter mates with the EKLF genotype indicated at the top of each lane. The RNA was analyzed by RNase protection using a human -globin and a
mouse -major probe. The RNA was also simultaneously analyzed with a
GATA-1 probe as a loading control. The position of the protected
fragments are indicated to the right of the figure.
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EKLF function is not developmental specific.
When the human -globin gene is alone in cis with the LCR, it
is expressed at all developmental stages.7,10,35 Thus, we
could determine whether the EKLF protein is active at early embryonic
stages by measuring the expression of the µD transgene in the
presence and absence of EKLF. RNA from yolk sacs (the site of primitive
erythropoiesis) was isolated from EKLF wild-type, heterozygous, and
null 10.5-day embryos. Human -globin-transgene expression was again
measured by RNase protection, but the embryonic stage-specific mouse
y-globin mRNA served as the control in this case.
Expression of the transgene was strongly dependent on EKLF. Very little
human -globin expression could be detected in
EKLF/ embryos, but the transgene was expressed at
approximately 30% the level of the mouse y gene in
wild-type and heterozygous embryos (Fig 4,compare lane 3 with lanes 1 and 2). Results identical to those shown in
Fig 4 were obtained for a second set of embryos in which two additional EKLF/ animals carried the transgene. Thus, the
dependence of -globin gene expression on EKLF, and hence EKLF
function, is not developmental specific.
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| Fig 4.
EKLF is also required for -globin-transgene
expression at the embryonic stage. RNA isolated from 10.5-day yolk sac
of embryos with the various EKLF genotypes and transgenicity for the
µD14 construct indicated at the top of the lanes was analyzed by
RNase protection with a human -globin-transgene probe and a mouse
 y-globin probe. The position of the protected fragments
detected by each probe is indicated by the arrows to the right of the
figure.
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DISCUSSION |
It had been suggested previously that EKLF function may be
developmental specific, partly because it is absent from K562 cells that express human embryonic and fetal globin genes, but not the adult
-globin gene.20 If this were the case, EKLF might serve as the predominant switch signal in the regulation of globin genes. Previously, it had been shown that EKLF mRNA and DNA-binding activity are indeed present in embryonic erythroid tissues.20,22,30 However, it had not been excluded that EKLF activity is controlled in a
developmental-specific manner. For example, EKLF function might depend
on protein modifications and/or protein partners that are
developmentally regulated. However, as reported here, we have found
that the -globin transgene in the µD construct is as dependent on
EKLF for expression in embryonic erythroid tissues as it is in fetal
liver (Fig 4). Thus, the function of EKLF is not developmental
specific. We conclude that EKLF cannot be responsible for the
initiation of globin gene switching, because it is present and fully
active before this process occurs. We cannot rule out that a protein
other than EKLF is involved in strengthening LCR
-globin-gene
interactions in a developmental-regulated fashion. However, expression
of the -globin gene at all developmental stages when present alone
with the LCR6,7 suggests this may not be the case.
Studies with several different transgene constructs have shown that the
-globin gene is downregulated in later development, at least partly,
irrespective of the presence of the -globin gene.6-9 We
predict, therefore, that it may be this autonomous downregulation of
the -globin genes that initiates the switch from -globin- to
-globin-gene expression. We suggest that the -globin gene is
fully primed to interact with the LCR well before it is actually
expressed. However, the interaction between the -globin gene and LCR
cannot occur until the -globin genes begin to be downregulated and
their interaction with the LCR is weakened. Once this occurs, the LCR
can switch from the -globin gene to the -globin gene. By
competing for LCR interactions in this manner, the -globin gene may
in turn help to downregulate the -globin genes more rapidly. In this
sense -globin downregulation appears to occur more slowly in both
heterozygous and homozygous EKLF knockout mice than in wild-type
animals, which results in increased -globin expression in fetal
life.15 This suggests that EKLF is involved in helping the
LCR change from the -globin to the -globin gene, presumably by
stabilizing LCR-globin-gene interactions.15 However,
our results show that active EKLF in itself is not sufficient to induce
the process. We therefore believe it is a combination of EKLF and the
initiation of the shutdown of the -globin genes that is needed for
the switch from -globin- to -globin-gene expression to occur at
the proper time and rate during development.
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FOOTNOTES |
Submitted November 19, 1997;
accepted January 5, 1998.
Supported by a grant from the Medical Research Council of Canada to
L.W., through a donation from the Fondation de l'Hôpital Notre-Dame, and by salary support from Défi corporatif Canderel to Q.M. L.W. is a Scholar of the Fonds de la Recherche en Santé du Québec.
Address reprint requests to Lee Wall, PhD, Centre de
recherche du Centre hospitalier de l'Université de
Montréal, Room 4605, Pavillon Notre-Dame, 1560 Sherbrooke East,
Montreal, Quebec, Canada H2L 4M1.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
We are grateful to R. Kothary for critical reading of the manuscript.
We thank Dr Tim Townes and Dr Jim Bieker for providing an EKLF clone
and Dr James Ellis for providing the µD14 transgenic mice.
| |
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Abstract
Introduction
Abstract
