From the Division of Experimental Hematology,
Departments of
Biochemistry and ¶ Immunology, St. Jude
Children's Research Hospital, Memphis, Tennessee 38105 and the
§ Rotary Bone Marrow Research Laboratory, Royal Melbourne
Hospital Research Foundation, Parkville, Victoria 3050, Australia
Received for publication, May 19, 2000, and in revised form, September 7, 2000
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ABSTRACT |
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The NTF-like family of transcription factors have
been implicated in developmental regulation in organisms as diverse as
Drosophila and man. The two mammalian members of this
family, CP2 (LBP-1c/LSF) and LBP-1a (NF2d9), are highly related
proteins sharing an overall amino acid identity of 72%. CP2, the best
characterized of these factors, is a ubiquitously expressed 66-kDa
protein that binds the regulatory regions of many diverse genes.
Consequently, a role for CP2 has been proposed in globin gene
expression, T-cell responses to mitogenic stimulation, and several
other cellular processes. To elucidate the in vivo role of
CP2, we have generated mice nullizygous for the CP2 allele. These
animals were born in a normal Mendelian distribution and displayed no
defects in growth, behavior, fertility, or development. Specifically,
no perturbation of hematopoietic differentiation, globin gene
expression, or immunological responses to T- and B-cell mitogenic
stimulation was observed. RNA and protein analysis confirmed that the
nullizygous mice expressed no full-length or truncated version of CP2.
Electrophoretic mobility shift assays with nuclear extracts from
multiple tissues demonstrated loss of CP2 DNA binding activity in the
Cellular diversity is generated by unique combinations of
transcription factors interacting to specify patterns of gene
expression in different cell types. This is particularly evident during
development where biochemical and genetic studies have identified
numerous proteins essential for the processes that govern
embryogenesis. Many of these factors are highly conserved in evolution,
playing critical roles in organisms as diverse as Drosophila
and man. One family of transcription factors that typifies these
principles is the NTF-like group of proteins. The founding member of
this family is the developmentally programmed Drosophila
factor, NTF-1 (neurogenic element binding
transcription factor) (1). NTF-1 (also known as
Grainyhead or Elf-1) was first identified through its ability to bind a
cis element critical for expression of the Dopa
decarboxylase gene (2, 3). Subsequently, NTF-1 was shown to bind
to promoters of other developmentally regulated genes including
Ultrabithorax, fushi tarazu, and
engrailed (2). NTF-1 has also been linked to dorsal/ventral
and terminal patterning through the formation of multiprotein complexes
that influence transcription from the decapentaplegic and
tailless genes (4, 5). More recently, tissue-specific
isoforms of the protein have been described in Drosophila,
and mutation of these isoforms or the ubiquitously expressed gene
results in pupal lethality with gross developmental defects (1, 6).
In mammals, two highly related NTF-like genes have been identified. In
humans they are known as LBP-1a and CP2 (LBP-1c/LSF), whereas the mouse
homologues are referred to as NF2d9 and CP2, respectively (7-10). The
human genes are 72% identical in overall amino acid sequence but share
higher sequence identity (88%) in the N-terminal halves of the
proteins than the C-terminal halves (52%). The homology with the NTF
gene is also in the N-terminal region with three amino acid stretches,
148 to 159, 205 to 216, and 233 to 246 showing 66, 75, and 79%
identity, respectively. The NTF-like gene family has been shown to have
a variety of cellular and developmental functions in human and murine
cells. The best characterized member of the family, CP2, was initially
identified as a factor that binds to, and stimulates transcription
from, the murine A developmental role for CP2 has been identified in studies of globin
gene regulation. In this context, CP2 binds to the stage selector
element in the proximal Despite the extensive literature examining CP2 function in
vitro and in cell lines, the in vivo role of this
factor remains unknown. To address this, we have generated a CP2 null
mutation in mice by homologous recombination. Mice lacking CP2
expression were examined for defects in growth and development, with a
particular emphasis on hematopoiesis, immune, and neural function. We
observed no significant abnormality in CP2 Generation of CP2 In Situ Hybridization Studies--
Embryo sections were prepared
as described previously (25). Briefly, C57/BL6 mice, overdosed with
ketamine and rhompin, were perfused intracardially with
paraformaldehyde, and the embryos from timed matings were postfixed in
a similar solution. Sections of 10-14 µM were cut with a
cryostat, mounted on glass slides, and stored at Phenotypic Analysis--
Tissues from normal and age-matched
heterozygote and homozygote knockout mice were removed and fixed in
formalin, and embedded paraffin sections were prepared. These sections
were stained with hematoxylin and eosin and examined by light
microscopy. Peripheral blood (150 µl) was obtained by retro-orbital
puncture and blood cell counts, and erythrocyte parameters were
determined utilizing an automated analyzer (Coulter). In addition an
aliquot was stained with Wright's Giemsa or methylene blue to study
hematopoietic cell morphology and reticulocytes, respectively. Bone
marrow hematopoietic progenitors were cultured in methylcellulose in
the presence of IL3, erythropoeitin, and stem cell factor (Terry
Fox Laboratories, Vancouver, Canada). For immunological studies, single
cell suspensions were prepared from spleen, lymph node, thymus, and
bone marrow and stained with cell type-specific markers for
granulocytes (Gr1), T-cells (CD8 and CD4), B-cells (B220 and
IAb), and NK cells (NK1.1). Fluorescence analysis
was performed utilizing a FACScan flow cytometer (Becton Dickinson,
Mountain View, CA). Similar cellular suspensions were utilized to
assess the proliferative potential of T- and B-cells, culturing
105 cells in the presence of anti-CD3 Analysis of Gene Expression by Ribonuclease Protection Assays
(RPA)--
RNA was prepared from various tissues and from peripheral
blood and bone marrow cells using RNAzol B or RNASTAT 60. Murine CP2
and NF2d9 cDNA fragments spanning exons 2 and 3 and a portion of
exon 4 were subcloned into pSP73. RPA studies were performed using the
Ambion RPAII kit as per the manufacturer's instructions. For studies
of murine and transgenic human globin gene expression RPA was performed
utilizing probes specific for the murine Nuclear Extract Preparation and Electrophoretic Mobility Shift
Assays (EMSA)--
Crude nuclear extracts were prepared from various
primary tissues (26, 27) and quantitated using the Bio-Rad protein
assay system as per the manufacturer's instructions. EMSAs were
performed by incubating varying amounts of nuclear extract with
105 cpm of [32P]dCTP end-labeled double
stranded oligonucleotides encoding the CCAAT box region of the murine
Yeast Two Hybrid Assays--
cDNA sequences encoding the
C-terminal 250 amino acids (amino acids 250-500) of CP2 and the
corresponding region of LBP-1a were inserted into the yeast expression
vector pGBT9. The resultant plasmids encode hybrid proteins containing
the DNA binding domain of GAL4 fused to CP2 or LBP-1a residues. The
yeast reporter strain, HF7C, was transformed with this vector and a
second plasmid encoding a hybrid protein of the GAL4 transactivation
domain and either the NF-E4 cDNA or RING1B cDNA. The yeast were
plated on leucine/tryptophan/histidine plates and incubated at 30 °C
for 3 days (28). Protein interactions are indicated by growth on these
plates. Transfection efficiency was monitored by plating of an aliquot
of the transformation on leucine/tryptophan plates (data not shown).
Transgenic Mice--
CP2 Targeting of the CP2 Genomic Locus--
To disrupt the murine CP2
gene, a targeting vector was designed that replaced the first
untranslated exon and the entire second exon containing the initiation
codon and the trans-activation domain with a hygromycin
expression cassette (Fig. 1A).
In addition, this cassette introduced termination codons in all open
reading frames. RW8 embryonic stem cells were electroporated with this construct and selected in hygromycin and FIAU. Southern analysis of
resistant clones demonstrated a 9.0-kb EcoRI fragment in
addition to the 10.5-kb wild type allele, at a mean frequency of one in 25 clones (data not shown). Four independently targeted clones, with
normal karyotypes, were injected into C57BL/6J blastocysts, three of
the clones being transmitted through the germ line. Interbreeding of
mice heterozygous for the CP2 allele (CP2+/ Expression of CP2 in Homozygous CP2 Phenotypic Analysis of CP2 Examination of Hematopoiesis in CP2
It is possible that despite normal adult erythropoiesis, the loss of
CP2 expression may affect either DNA Binding Activity in Extracts from CP2
The lack of an obvious phenotype in the CP2 Expression of CP2 and NF2d9 during Mouse Development--
To
determine whether the distribution of expression of CP2 and NF2d9 was
similar, we performed in situ hybridization on embryo sections using antisense and sense probes specific for each mRNA transcript. Normal embryos were examined at E9.5, E11.5, and E13.5 dpc.
Probes were of similar specific activity, and sense probes produced
little background signal from embryos probed at all developmental stages (Fig. 4, A and
C). However, utilizing an antisense probe, we observed CP2
expression in most tissues at similar levels at E13.5 dpc (Fig.
4B). In contrast, although expression of NF2d9 was observed
in all tissues, it was markedly higher in the fetal liver (Fig.
4D, arrow). It is possible that loss of CP2
expression results in up-regulation of NF2d9 expression. To test
this hypothesis, we determined the expression of NF2d9 in the
brain, kidney, and heart of CP2+/+ and CP2 null animals. As
shown in Fig. 4E, no significant change in the relative
expression of NF2d9 was observed in any of these tissues when compared
with the actin control.
LBP-1a Can Functionally Replace CP2 Activity--
The DNA binding,
transactivation, and expression patterns of LBP-1a/NF2d9 suggested that
this protein could potentially compensate for the loss of CP2. To
evaluate this further, we examined whether LBP-1a could fulfill the
protein-protein interaction role of CP2. We have recently defined two
transcription factors that specifically interact with
CP2.4 The first, NF-E4, is
the fetal/erythroid-specific component of the SSP. The second, RING1B,
is a RING finger domain-containing protein involved in the regulation
of CP2-dependent transcription. Utilizing the yeast two
hybrid assay system we compared the ability of CP2 and LBP-1a to
interact with these known CP2 partners. As shown in Fig.
5A, growth on
leucine/tryptophan/histidine plates, which is indicative of a
protein-protein interaction, was observed with both CP2 and LBP-1a and
NF-E4. Protein interactions were also observed between CP2 and LBP-1a
and RING1B (Fig. 5B). No growth was observed with controls
lacking either of the interacting proteins.
Prompted by studies documenting the importance of the NTF-1 gene
in Drosophila development, we have examined the effects of gene targeting of the mammalian NTF-like gene, CP2, in mice. These experiments assumed additional importance with the identification of
CP2 as a major component of the SSP, a protein complex involved in the
regulation of fetal hematopoiesis and with the identification of CP2 as
a key factor in the T-cell proliferative response (17, 19). To our
surprise, no difference in hematopoiesis, globin chain synthesis, or
immunological function between wild type and CP2 null animals was
observed. Indeed, the general physiology, behavior, and reproductive
capacity of CP2 Although our data is consistent with redundancy of function in the
mammalian NTF-like gene family, it was essential to rule out the
possibility that the knockout phenotype was masked by the production of
a truncated or alternately spliced form of CP2. Several lines of
evidence suggest that this did not occur. First, RNase protection and
RT polymerase chain reaction analysis failed to show evidence of either
the 5' exons 1 and 2 or the 3' end of the coding sequence. Second,
DNA-protein interaction studies demonstrated loss of the CP2
homodimeric band with the appearance of a slower migrating complex,
which we attributed to NF2d9. The ability of LBP-1a/NF2d9 to bind to
CP2 consensus sites is not surprising. Examination of the amino acid
sequence of the respective proteins reveals striking homology in the
region that we, and others, have identified as the DNA binding domain
(18, 30).5 Between residues
150 and 291, the core of the binding domain, the two proteins share
90% identity and 96% similarity. Previous studies have demonstrated
that LBP-1a and CP2 can bind the CP2 consensus sequence adjacent to the
HIV initiation site (9). We have extended that observation, confirming
that LBP-1a also binds to the CP2 sites in the SV40 major late promoter
and the murine The migration pattern we observed with CP2 The ability of LBP-1a/NF2d9 to fully compensate for CP2 loss in the
context of heteromeric protein interactions was less assured. Sequence
comparison of the previously characterized dimerization domain of CP2
reveals that it shares 52% identity and 75% similarity with LBP-1a at
amino acid level. Recently, the dimerization domain of CP2 has been
refined to a region between residues 266 and 403 (18). In this
sequence, the two proteins share 63% amino acid homology and 85%
amino acid similarity. Therefore it was not surprising that
protein-protein interactions between LBP-1a and previously identified
partners of CP2 were conserved.
Our studies of gene expression lend further support to our hypothesis
that LBP-1a/NF2d9 rescues CP2 nullizygous animals. In situ
hybridization analysis revealed that CP2 and NF2d9 are widely co-expressed, albeit at differing levels. One striking difference was
observed in the pattern of expression between the two genes, with NF2d9
being present at significantly higher levels in the fetal liver. Two
interpretations of this result are suggested by the known roles of CP2
and NF2d9. First, NF2d9 has been linked to gender-specific expression
of the steroid 16 The ability of one highly related gene to compensate for the loss of
another in gene-targeting experiments is widely recognized. Redundancy
may be observed for all functions of the protein or may be limited to a
single organ system (32, 33). Studies of the maf transcriptional
regulators highlight the complexity of gene-targeting analysis in a
multigene family. Although two of the family members, mafK and mafG,
share overall 62% amino acid identity, with higher degrees of homology
in known functional domains, the phenotype of nullizygous animals is
widely divergent (34). Unlike the normal phenotype observed with
mafK-deficient animals, mafG nullizygotes display impaired
megakaryocytic development and behavioral defects. This divergence
occurs despite largely identical patterns of expression.
Despite the evidence suggesting that NF2d9 can fully compensate for the
loss of CP2 in vivo, it is possible that a subtle CP2-specific phenotype exists in the nullizygous mice. For example, although we have performed extensive phenotypic analysis of this strain, it is possible that we have not identified the tissues or
tissues that require study. For example, we have closely evaluated the
histological features of the central nervous system in the CP2/
lines. However, a slower migrating complex that was ablated with
antiserum to NF2d9, the murine homologue of LBP-1a, was observed with
these extracts. Furthermore, we demonstrate that recombinant LBP-1a can
bind to known CP2 consensus sites and form protein complexes with
previously defined heteromeric partners of CP2. These results suggest
that LBP-1a/NF2d9 may compensate for loss of CP2 expression in
vivo and that further analysis of the role of the NTF family of
proteins requires the targeting of the NF2d9 gene.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-fibrinogen promoter and the viral SV40 major late promoter (11, 12). Binding sites for CP2 have also been defined in
regulatory regions of the human immunodeficiency virus
(HIV)1 where it acts in
concert with YY1 to repress transcription (9, 13-16). In the context
of non-viral gene regulation, CP2 has been shown to bind homomerically
to the human c-fos, ornithine decarboxylase, c-myc, and DNA polymerase promoters and the murine
-globin and fibrinogen promoters and activate transcription in
vitro (17, 18). Binding to the regulatory elements in the
fos and ornithine decarboxylase promoters is modulated by
cell growth signals. Mitogenic stimulation of resting T-cells is
associated with rapid phosphorylation of CP2 by the mitogen-activated
protein kinase pp44 (extracellular signal-regulated kinase 1) and a
consequent increase in its DNA binding activity (17). This modulation
suggests that CP2 contributes to the regulation of early response genes
and therefore plays a role as a cell growth regulator.
-gene promoter as a heteromeric complex with
a recently cloned fetal/erythroid-specific partner protein, NF-E4
(19).2 This complex,
known as the stage selector protein (SSP), contributes to the
preferential recruitment of the
-globin locus control region to the
-promoter during fetal erythropoiesis (20, 21). SSP binding sites
have also been defined in the
-promoter and in the regions of DNase1
hypersensitivity that constitute the locus control region (19, 22,
23).
/
mice
compared with wild type littermates. We have shown through DNA binding
and protein-protein interaction studies that the lack of a discernible
phenotype may be due to a complete rescue by NF2d9, the murine
homologue of LBP1a.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
Mice--
We
isolated eight CP2 genomic clones by screening a 129-derived ES
cell phage library with a full-length mouse CP2 cDNA probe. Duplicate lifts screened with a probe specific for exon two, containing the initiation ATG, identified one clone with a 12-kb insert that encodes the first two exons and the 5' untranslated region. Detailed restriction endonuclease mapping of this fragment confirmed the previously reported genomic structure of murine CP2 with the exception of an EcoRI polymorphism detected in the 5' untranslated
region (see Fig. 1A) (24). Subsequently, a 6.6-kb
NcoI fragment containing the 5' untranslated region and a
portion of the first exon was subcloned into pSL301. A
XhoI-SalI fragment containing a phosphoglycerate kinase promoter-regulated hygromycin resistance expression cassette was
cloned into a downstream SalI site. Finally, a 3.4-kb
SalI-NotI fragment containing 2.4 kb of the
second intron of CP2 and a 1-kb HSV-TK expression cassette
fragment (a kind gift of Dr. J. vanDeursen) was cloned downstream of
this region to provide 3' homology and a negative selectable marker.
This construct, pK01HygTK, was linearized with NotI and
transfected by electroporation into RW8 embryonic stem cells (Genomic
Systems Inc). The cells were cultured on primary irradiated embryonic
STO feeder cells in the presence of 140 fg/ml hygromycin and 0.2 fM FIAU. Resistant
clones were screened by Southern blot analysis using a unique 0.5-kb
SalI-NcoI fragment located 5' to the targeted
sequence. Four karyotypically normal CP2+/
clones were
microinjected into C57BL/6 blastocysts, of which three clones were
transmitted through the germ line.
20 °C. These
slides were subsequently probed with sense and antisense riboprobes
generated by [33P]UTP labeling from Bluescript plasmids
encoding the complete cDNAs of CP2 and NF2d9 (the latter were a
kind gift of Dr. M. Negishi) (10). Specific signals were developed by
dipping the slides in NTB2 emulsion (Kodak Scientific Imaging
Systems) and exposed at 4 °C for two weeks. The sections were
counterstained using 0.1% toluidine blue in distilled water and
analyzed by phase-contrast microscopy.
+/
phorbol
12-myristate 13-acetate, concanavalin A, lipopolysaccharide,
phytohemagluttinin, ionomycin, or a combination of phorbol 12-myristate
13-acetate, phytohemagluttinin, and ionomycin for 48 h as
described previously (17).
,
,
y,
h1, and
major transcripts and the human
-, G
-,
and
-globin transcripts (a kind gift of Dr. K. Gaensler).
-globin promoter, the
-fibrinogen promoter, or the SV40 major
late promoter in a 20-µl reaction containing 500 ng of
poly[d(I·C)], 6 mM MgCl2, 16.5 mM KCl, and 100 µg of bovine serum albumin (21,
27). For antibody studies, 3 µl of preimmune serum or rabbit
anti-mouse CP2 antibody were preincubated for 10 min with the binding
reaction prior to addition of the probe. Polyclonal antiserum against
NF2d9 was kindly provided by Dr. M. Negishi. 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. Recombinant CP2 and
NF2d9 were prepared as glutathione S-transferase fusion
proteins as described previously (19).
/
mice were bred with
mice transgenic for a single copy of a yeast artificial chromosome
(YAC) containing 250 kb of the human
-globin locus (
-YAC) (a kind
gift of Karin Gaensler, University of California, San Francisco,
CA) (29). Timed pregnancies were set up utilizing
CP2+/
females and CP2+/
YAC+
males, where the day of plug formation was taken as 0.5 days post-coitum (E0.5 dpc). Embryos were collected on days E9.5 dpc, E10.5
dpc, E11.5 dpc, and E14.5 dpc and genotyped by standard methodologies
utilizing the CP2 probes described above and a probe specific for
IVSII of the human A
-globin gene (a kind gift of
Dr. Karin Gaensler). RNA from yolk sac and fetal liver was prepared,
and RPA analysis was performed as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) resulted in
litters of normal size with the expected Mendelian frequency of
genotypes. Of 256 total offspring tested, 72 were CP2+/+
(28%), 125 were CP2+/
(49%), and 60 animals (23%) were
nullizygous (CP2
/
) for the CP2 allele.
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Fig. 1.
Generation of CP2 null mice.
A, strategy for targeted disruption of the CP2 gene. The
upper map shows a 12-kb fragment encoding the 5' end of the
murine CP2 locus. The middle map demonstrates the
targeting construct, pK01HygTK, a hygromycin expression cassette
replacing the 3' portion of exon 1 and all of exon 2. A 3' HSV-TK
cassette allows for negative selection of hygromycin-resistant clones
utilizing FIAU. The lower map delineates the disrupted
allele. Homologous recombinants were identified utilizing a unique 5'
probe (hatched box). The sizes of the wild type and
disrupted alleles detected by this probe are indicated. B,
RNase protection analysis of total RNA isolated from multiple tissues
of CP2+/+ and CP2 /
mice. Total RNA was
extracted from brain (B), heart (H), kidney
(K), lung (L), and spleen (S) of
CP2+/+ and CP2
/
animals or from the murine
erythroleukemia cell line (MEL). RNA was hybridized to a
mixture of CP2- and actin-specific probes adjusted to equal specific
activities. Protected fragments are indicated at the right.
This experiment is representative of 10 animals assayed.
Dashes indicate empty lanes.
/
Animals--
To confirm the loss of CP2 gene expression in nullizygous
animals, RNA was prepared from various tissues of both wild type and
CP2
/
mice and analyzed by RNase protection analysis. A
specific band of 380 nucleotides was observed in all tissues derived
from wild type animals utilizing a riboprobe that hybridizes to exons
2-4 (Fig. 1B, lanes 1-5). In contrast, no
signal was observed utilizing RNA derived from the brain, heart,
kidney, lung, and spleen of CP2
/
mice (Fig.
1B, lanes 7-11). An actin probe controlled for
the integrity of the RNA (Fig. 1C). To confirm the loss of
CP2 expression, and to rule out a cryptic splicing event that might
produce a functional CP2 transcript, RNA from wild type and
CP2
/
tissues was assayed by RT polymerase chain
reaction utilizing primers specific to the 3' end of the mRNA
transcript. Although a CP2-specific signal was observed in all wild
type tissues tested, no signal was detected from RNA derived from
CP2
/
animals (data not shown).
/
Animals--
Male and
female knockout mice grew normally and were healthy up to 18 months of
age. No abnormal behavioral patterns were observed. The fertility of
CP2
/
animals was normal, and no increase in morbidity
was observed when compared with littermate controls. Careful
histopathological examination of brain, spleen, kidney, liver, thymus,
lymph nodes, heart, skin, muscle, and bone from CP2
/
animals, performed at 3, 9, and 15 months, was identical to wild type
littermate controls (data not shown).
/
Animals--
CP2 has been implicated in the regulation of several
hematopoietic genes, particularly those of the globin loci (7, 19). To
determine whether loss of CP2 expression resulted in changes in
hematopoiesis, the hematological parameters of CP2
/
mice were assayed and compared with those of wild type littermates. No
significant difference in total cell counts, hematocrits,
reticulocytes, differential white cell counts, or the
-/
-globin
ratio was observed (Table I). In
addition, the numbers of bone marrow progenitors, as measured by
colony-forming unit activity, were similar in CP2+/+ and
CP2
/
animals (data not shown). Similar studies of
lymphopoiesis were stimulated by recent studies implicating CP2 in the
modulation of T-cell proliferative responses (17). However, extensive
analysis of T, B, and NK phenotypes, as well as functional assays of B- and T-cell function, failed to identify a difference between
CP2+/+ and CP2
/
cells (data not shown).
Hematological analysis of 18 CP2+/+ and 18 CP2/
animals
- or
-globin gene expression
during hematopoietic ontogeny. CP2 was initially identified as an
-globin CCAAT box binding activity, suggesting a possible role in
-globin gene expression. However, neither
- nor
-globin gene
expression was perturbed in yolk sac or fetal liver cells (Fig.
2A). We have demonstrated that
CP2 is a component of the
-globin promoter-binding SSP complex and
suggested that the
to
switch in the
-globin subtype may be
perturbed in a CP2 null environment. To test this hypothesis, we bred
CP2
/
animals with mice transgenic for a 240-kb YAC
containing the human
-globin locus (
YAC). Subsequently, we bred
male progeny transgenic for the
YAC with CP2+/
females
and examined the expression of both human and mouse
-globin-like genes at several developmental stages. As shown in Fig. 2B,
both human and murine
-globin-like gene expression in yolk sac,
fetal liver, and bone marrow were identical in CP2+/
and
CP2
/
embryos and adult mice, respectively.
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Fig. 2.
Effects of loss of CP2 expression on globin
gene expression. A, analysis of murine -like globin
gene expression. Total RNA was extracted from yolk sacs (E9.5
dpc) and fetal liver (E14.5 dpc) of wild type
(CP2+/
) or CP2
/
animals and hybridized to
a mixture of murine
-globin,
-globin, and actin riboprobes. This
experiment is representative of several littermates assayed.
B, analysis of murine and human
-globin-like gene
expression in mice transgenic for the
-globin locus YAC. Total RNA
was extracted from yolk sacs (E9.5 dpc), fetal liver
(E14.5 dpc), or adult bone marrow (Adult) of
heterozygote (CP2+/
) or CP2
/
animals
transgenic for the human
-globin YAC and hybridized to a mixture of
human
-globin, murine
H1-globin, and actin riboprobes (d9.5) or
human
-globin, human
-globin, murine
major-globin,
and actin riboprobes (d14.5 and adult). Protected fragments are
indicated. Littermates that do not contain the
-globin YAC are
included for comparison. This experiment is representative of several
litters assayed.
/
Mice--
To examine CP2 DNA binding site occupancy in the null mice,
we prepared crude nuclear extracts from lung, kidney, heart, and liver
of wild type and CP2
/
animals and performed EMSA using a double
stranded oligonucleotide probe containing the
-globin CCAAT box (7).
Utilizing equal amounts of protein in each lane, a band of similar
electrophoretic mobility was observed in all wild type tissues (Fig.
3A, compare lanes
1, 3, 5, and 7). In contrast,
extracts from CP2
/
tissues failed to show the band seen
with wild type extract and instead showed a DNA-protein complex with a
slower migration pattern (Fig. 3A, compare lanes
2, 4, 6, and 8 with 1,
3, 5, and 7, respectively). This
result was not dependent on the amount of protein added, as 2- to
8-fold more protein from CP2
/
liver extract incubated
with the probe generated an identical band shift (Fig. 3A,
compare lane 7 with lanes 8-11).
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Fig. 3.
A, analysis of protein/DNA
binding activity in nuclear extracts from CP2+/+ and
CP2 /
tissues. Equal amounts of protein derived from
nuclear extracts from lung, kidney, heart, and liver of
CP2+/+ and CP2
/
animals (lanes
1-8) were incubated with an
-globin CCAAT box CP2 binding
probe. Both the CP2 complex (CP2) and a slower migrating
complex (*) are indicated at right. In
lanes 9-11, 2-, 5-, and 10-fold more extract was incubated
with the probe compared with lanes 7 and 8.
Similar results were observed with a
-fibrinogen probe (data not
shown). B, recombinant CP2 and NF2d9 have differing
electrophoretic mobilities. Equal amounts of recombinant protein were
incubated with an
-globin CCAAT box CP2 binding probe. Both the CP2
complex (CP2) and the NF2d9 complex are indicated.
Similar results were observed with a
-fibrinogen probe (data not
shown). C, effect of NF2d9-specific antisera on EMSA.
Nuclear extracts derived from livers of CP2+/+ (lanes
1 and 3) or CP2
/
(lanes 2 and 4) mice were incubated with an
-globin CCAAT box CP2
binding probe in the presence of 1 µl of NF2d9-specific monoclonal
antiserum (lanes 3 and 4). An approximately 5-fold excess of
nuclear extract was used in lanes 2 and 4 in
contrast with lanes 1 and 2.
/
animals
coupled with the persistent DNA site occupancy observed with extracts from nullizygous tissues suggested the presence of a ubiquitous CP2-like factor that could compensate for the lack of CP2. One candidate factor was Nf2d9, the murine homologue of the human NTF-like gene, LBP-1a (10). Support for this hypothesis was obtained by
studying the relative electrophoretic mobilities of CP2 and NF2d9. Both
molecules bound the
-globin CCAAT box and
-fibrogen probes, the
NF2d9-DNA complex having a perceptibly slower mobility (Fig.
3B and data not shown). To determine whether the protein-DNA
complex generated with CP2
/
extracts contained NF2d9,
we performed competition experiments utilizing excess concentrations of
unlabeled oligonucleotides that have been previously shown to bind CP2
and/or NF2d9(7, 10). These oligonucleotides were capable of ablating
both wild type and mutant binding activity (data not shown). We also
investigated the ability of monoclonal antisera generated against
recombinant NF2d9 to disrupt binding activity. This antisera does not
cross-react with CP2 as assessed by
immunoblotting.3 Addition of
the antibody induced a partial supershift of wild type binding activity
(Fig. 3C, compare lanes 1 and 3). In
contrast, mutant activity was completely supershifted (Fig.
3C, compare lanes 2 and 4). These data
suggest that NF2d9 can maintain DNA site occupancy at CP2 binding sites.
View larger version (77K):
[in a new window]
Fig. 4.
Expression pattern of CP2 and NF2d9 in wild
type and mutant embryos. A D, sequential sagittal
sections of wild type murine embryos E13.5 dpc were subjected to
in situ hybridization with either CP2 (A) or
NF2d9 (C) 33P-labeled sense riboprobes or CP2
(B) or NF2d9 (D) antisense riboprobes.
E, analysis of NF2d9 expression in CP2+/+ and
CP2 null animals. Total RNA was extracted from brain, heart, and kidney
of wild type (CP2+/+) or CP2
/
animals and
hybridized to a mixture of human NF2d9 and actin riboprobes (d9.5).
Protected fragments are indicated. This experiment is representative of
several litters assayed.
View larger version (80K):
[in a new window]
Fig. 5.
LBP-1a interacts specifically with CP2
interacting proteins, NF-E4, and Ring1B. A, yeast two
hybrid assay of CP2/LBP-1a and NF-E4. The Saccharomyces
cerevisiae reporter strain HF7C was transformed with the indicated
plasmids. pGB-LBP-1a and pGB-CP2 contain the dimerization domains of
LBP-1a and CP2, respectively, fused in frame with the DNA binding
domain of GAL4 (amino acids 1-147). pACT-NF-E4 contains the entire
coding sequence of the NF-E4 cDNA fused in frame with the
activation domain of GAL4 (amino acids 768-881). A specific
interaction between pTD encoding the SV40 T-antigen and pVA3 encoding
p53 has been reported previously. Yeast transformants were streaked
onto synthetic media plates lacking leucine, tryptophan, and
histidine (LTH ) to assess potential protein
interactions. B, yeast two hybrid assay of CP2/LBP-1a and
Ring1B. The experiments were performed using the above methodology with
the exception that pACT-Ring1B was substituted for pACT-NF-E4.
pACT-Ring1B contains the entire coding region of the Ring1B cDNA
fused in frame with the activation domain of GAL4 (amino acids
768-881).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
animals was identical to wild type
littermates. Examination of the binding activities of nuclear extracts
suggested that CP2 consensus binding sites are occupied in
CP2
/
animals by NF2d9, a protein highly related to CP2.
In addition, we have shown that NF-E4 and RING1B, known heteromeric
partners of CP2, also form protein complexes with LBP-1a/NF2d9. The
similar patterns of expression of the two highly related genes coupled with the DNA and protein binding data suggests that the lack of a
discernible phenotype in the CP2 nullizygous mice may be due to rescue
by NF2d9.
-fibrinogen and
-globin promoters. These sequences
are archetypal CP2 binding sites in that they consist of a pair of direct repeats (G/A)CTGG spaced by an intervening sequence of variable
content, but set length, which restricts protein binding to a single
face of the DNA helix (18, 27). It is therefore likely that the other
target sites for CP2 will also allow binding of LBP-1a.
/
extracts in
the EMSA was consistent with a protein-DNA complex containing NF2d9. The slightly slower migration reflects the larger size of the NF2d9
protein and is consistent with previous reports and our observations of
the difference in the migration of recombinant CP2 and NF2d9 (Fig.
3B) (9). The ability of antiserum that recognizes only NF2d9
to displace the complex observed in CP2
/
animals,
coupled with the partial displacement observed with wild type extract,
further supports this conclusion. Previous studies and the results
reported here demonstrate the ability of LBP-1a/NF2d9 to functionally
compensate for CP2 in its transcriptional roles (9). The activation
domains of the two proteins have been mapped by our group to the
N-terminal 40 amino acids. In this region, the two factors are 88%
identical. We and others have demonstrated transcriptional activity of
both CP2 and LBP-1a/NF2d9 in yeast and mammalian cells and in in
vitro transcription assays (9, 15, 17,
30).6
-hydroxylase P450 (Cy2d-9) gene in the mouse liver
(10). In this setting, NF2d9 forms a heteromeric complex with an
unknown partner protein. Enrichment of NF2d9 in this organ may indicate
that it is the preferred protein partner for this developmental process
for reasons that are as yet unknown. Second, the fetal liver is a site
of hematopoiesis in the developing mouse. Studies from mice transgenic
for the human
-globin locus YAC demonstrate that a distinct fetal
stage of human
-gene expression occurs between days 10.5 and 13.5 (29, 31). As the SSP is involved in the preferential expression of this
gene during fetal erythropoiesis it is conceivable that NF2d9 and not
CP2 is the primary partner of NF-E4 in the formation of this complex
(21). Studies of human globin chain synthesis in NF2d9 nullizygous mice
will address this question.
/
animals, as well as their behavioral patterns in
view of the variations in patterns of expression in various parts of
the developing brain. Although we have observed no abnormality, it is
possible that an abnormal phenotype may become evident with the
establishment of the CP2 null genotype in different inbred strains. Our
studies documenting the pattern of expression of CP2 and NF2d9 failed to provide clues as to possible organs in which CP2 may be
non-redundant. However, they did suggest that NF2d9 may play a key role
in fetal liver function. The generation of NF2d9-deficient animals and their interbreeding with the CP2 nullizygous mice will address this issue.
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ACKNOWLEDGEMENTS |
---|
We thank Ngoc Tran, Amy McEwen, and Anna Becher for technical assistance, Gerard Grosveld and Jan van Duersen for assistance with generation of targeted animals, and Frederique Zindy for assistance with in situ studies. We thank members of the Jane and Cunningham laboratories for helpful discussions and A. W. Nienhuis for continuing support.
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FOOTNOTES |
---|
* This work was supported by the National Health and Medical Research Council of Australia, The Wellcome Trust (to S. M. J.), the Anti-cancer Council of Victoria (to D. R. C.), National Institutes of Health Grant PO1 HL53749, Cancer Center Support CORE Grant P30 CA 21765, the American Lebanese Syrian Associated Charities, the Assisi Foundation of Memphis, the Cooley's Anemia Foundation (to L. R.), and the British Society for Hematology (to V. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Division of Experimental Hematology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Tel.: 901-495-2733; Fax: 901-495-2176; E-mail: john.cunningham@stjude.org.
Published, JBC Papers in Press, September 19, 2000, DOI 10.1074/jbc.M004351200
2 J. M. C. and S. M. J., submitted for publication.
3 A. T., J. M. C., and S. M. J., unpublished information.
4 S. M. J. and J. M. C., submitted for publication.
5 J. M. C. and S. M. J., submitted for publication.
6 J. M. C. and S. M. J., submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HIV, human immunodeficiency virus; SSP, stage selector protein; kb, kilobase pair; RPA(s), ribonuclease protection assay(s); EMSA(s), electrophoretic mobility shift assay(s); YAC, yeast artificial chromosome; dpc, days post-coitum.
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