By
From the * Department of Oncology and the Department of Experimental Pathology, Bristol-Myers
Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000
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Abstract |
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The transcription factor NF-B is sequestered in the cytoplasm by the inhibitor proteins of the
I
B family. Each member of the I
B exhibits structural and biochemical similarities as well as
differences. In an effort to address the functional redundancy of two closely related I
B molecules, I
B
and I
B
, we generated knock-in mice by replacing the I
B
gene with the I
B
gene. The knock-in mice do not express I
B
, but express a T7-tagged I
B
under the promoter and regulatory sequence of ikba. Unlike the I
B
-deficient mice, which display severe
postnatal developmental defects and die by postnatal day 8, homozygous knock-in mice survive to adulthood, are fertile, and exhibit no apparent abnormalities. Furthermore, thymocytes and
embryonic fibroblasts from the knock-in animals exhibit an inducible NF-
B response similar
to that of wild-type animals. These results indicate that I
B
and I
B
share significant similarities in their biochemical activity, and that they acquired their different functions from divergent expression patterns during evolution.
![]() |
Introduction |
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Nuclear factor B (NF-
B)1 plays an important role in
regulating genes involved in inflammatory and immune responses. In vertebrates, NF-
B consists of homo-
or heterodimers of the subunits p50, p52, RelA, RelB, and
cRel. These subunits share a highly conserved NH2-terminal sequence termed the Rel-homology domain (RHD),
which is required for DNA binding, dimerization, nuclear
localization, and interaction with the inhibitor I
B molecules (for review see references 1). In resting cells, NF-
B is sequestered in the cytoplasm by the inhibitor I
Bs.
Cytoplasmic retention is achieved via interaction between
a conserved sequence motif known as the ankyrin repeats
in the I
Bs and the RHDs of NF-
B subunits. Upon cell
stimulation by a variety of agents, specific serine residues on the I
Bs are phosphorylated, signaling for ubiquitination which in turn targets I
Bs for proteasome-mediated
degradation. NF-
B released from the inhibitor translocates into the nucleus and activates transcription of target
genes, which include those involved in inflammatory, immune, and acute phase responses.
In mammalian cells, the IB family consists of I
B
,
I
B
, I
B
, Bcl-3, p105, and p100 (for review see references 2). Knockout mice studies have revealed the critical roles that some of these I
B molecules play in development and immune responses. For example, the knockout
mice of I
B
have increased basal NF-
B activity in hematopoietic organs. Extensive granulopoeisis, dermatitis, and death by postnatal day 8-10 are observed in these mutant animals (5, 6). In contrast, p100-deficient mice displayed gastric hyperplasia and an impaired proliferative response in lymphocytes (7). Bcl-3-deficient mice develop
normally but are incapable of antigen-specific antibody response (8, 9). These differences in phenotypes suggest that
each I
B family member plays a unique and nonredundant
role in regulating NF-
B activity in the hematopoietic system.
Among the IB members, I
B
and I
B
are the most
prominent and have been extensively characterized. I
B
and I
B
were first identified as two fractions from HeLa
extracts that had similar kinetics of activities (10). In addition to the ankyrin motif, I
B
and I
B
also have similar
phosphoacceptor sites in their NH2 termini that mediate
signal-induced degradation and a similar specificity of interaction with the RelA and cRel complexes (11, 12).
Given these similarities, it seemed surprising that I
B
could not compensate for I
B
in the I
B
-deficient
mice. Two potential explanations are: first, in spite of the
similarities in structure, these two proteins have different in
vivo biochemical activities and, therefore, they cannot replace each other's function. Alternatively, they are in vivo
biochemically equivalent, but differential expression patterns have made them functionally incapable of compensating for one another. In an attempt to discriminate between
these two possibilities, we have replaced I
B
with I
B
using homologous recombination in embryonic stem cells
in order to determine whether I
B
can be functionally
replaced by I
B
. The targeting event brought the integrated I
B
gene under the control of the ikba promoter
and at the same time introduced a null mutation in ikba.
Here we report that the "knock-in" mice develop to adulthood without apparent abnormalities, are fertile, have no increase in their basal NF-
B activity, and can elicit NF-
B
responses. These observations contrast strongly with those obtained with the I
B
-deficient mice, which present extensive granulopoeisis, dermatitis, and death by postnatal day
8-10 (5, 6). Thus, our data demonstrate that in vivo I
B
can be functionally replaced by I
B
, and that these two
molecules have acquired different functions by differential
tissue pattern expressions and responses to NF-
B inducers.
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Materials and Methods |
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Generation of the IB
Knock-in Mice.
Western Blot Analysis, Electrophoretic Mobility Shift Assay, and Immunoprecipitation.
Single cell suspensions of thymocytes or splenocytes were prepared from 4-6-wk-old mice according to standard procedures (14) in RPMI 1640 containing 10% heat-inactivated FCS. Cells were incubated with 20 ng/ml of PMA and 1 µg/ml of PHA, or 5 ng/ml mouse TNF at 37°C for the indicated periods of time before harvest. Preparation of cytoplasmic and nuclear extracts was performed as previously described (15). For immunoprecipitation, a total of 5 × 105 ES cells or 6 × 106 thymocytes were incubated with 0.5 mCi/ml of [35S]methionine in methionine-free DMEM containing 10% dialyzed FCS for 8 h at 37°C. Cells were lyzed in radioimmunoprecipitation assay buffer and immunoprecipitation was performed as previously described (16). Western blot analysis using equal amounts of cytoplasmic extracts was carried out using standard protocols. For electrophoretic mobility shift assay (EMSA), nuclear extracts were tested for binding to the palindromicHistology and Flow Cytometry Analysis.
Mouse tissues were immersion fixed in 10% buffered formalin and embedded in paraffin blocks. Sections were stained with hematoxylin and eosin. Flow cytometry analysis with single cell suspension from thymus, spleen, and bone marrow was performed using commercially available antibodies with a flow cytometer (Becton Dickinson, San Jose, CA). 3 × 105 cells were first incubated with 1 µg of the antibodies and then incubated with PE- or FITC-conjugated antibodies. ![]() |
Results |
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The murine IB
gene was replaced by the I
B
gene in ES cells by homologous recombination with the gene-targeting vector pPNT-abki (Fig. 1
A). The targeting vector deleted the entire coding sequence
of the I
B
gene and replaced it with the genomic sequence encoding the murine I
B
gene. To distinguish the
I
B
introduced by homologous recombination from the
endogenous I
B
, a 34-bp nucleotide sequence encoding
the bacterial phage T7-Tag was placed in front of the ATG
start codon of the I
B
gene in the targeting vector. This
targeting vector was transfected into CJ7 ES cells, and ES
cell clones carrying the replacement of the endogenous
I
B
gene were identified by Southern blot analysis (Fig. 1
B). The resulting knock-in allele was abbreviated as +/ki or
ki/ki for heterozygotes or homozygotes, respectively.
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To ensure that the homologous recombination resulted
in replacement of the IB
gene with a functional I
B
gene, an immunoprecipitation experiment was performed
in the ES cells. Extracts from [35S]methionine labeled wild-type (+/+) and recombined (+/ki) ES cells were immunoprecipitated with antiserum against I
B
(Fig. 1 C, lanes
1 and 2), and then with antiserum against the T7-Tag epitope (Fig. 1 C, lanes 3 and 4). The T7-Tag antiserum
precipitated a protein that is similar in molecular weight to
the I
B
protein in the recombinant ES cells but not in the
wild-type cells, indicating that the introduced I
B
was
expressed and can be distinguished from the endogenous
I
B
using antibodies against the T7-Tag epitope.
Aggregation chimeras giving
germline transmission were obtained from two targeted ES
cell lines. Both lines of heterozygous animals (+/ki) were
crossed to obtain homozygous mice (ki/ki). Homozygous
ki/ki mice were born in normal Mendelian ratio, indicating that replacement of IB
with I
B
did not affect embryonic development (Fig. 1 D).
In contrast to the previously described IB
-deficient
mice, which have severe developmental defects and die by
postnatal day 8 (5, 6), both lines of the ki/ki mice developed to adulthood. Examination of 5-wk-old ki/ki mice
revealed that the phenotype was both grossly and histologically similar to that of wild-type mice. Additionally, peripheral blood cells and serum biochemical analysis were
also similar to those from wild-type animals. In particular,
there was no evidence of granulopoiesis or epidermal dysplasia with hyperkeratosis as reported in the I
B
-deficient
mice (data not shown). The lack of granulopoiesis was further confirmed by flow cytometric analysis of bone marrow from wild-type and knock-in animals using the granulocyte/macrophage-specific surface markers Mac-1 and Gr-1.
Additionally, a normal distribution of B and T lymphocyte
markers (CD4, CD8, TCRab, Thy1.2, CD25, B220, and
IgM) and erythroid marker (Ter119) was observed, indicating that the lymphoid and erythroid compartments of these
animals are also normal (data not shown).
To determine if replacement of IB
by I
B
affected the
overall patterns of I
B and NF-
B gene expression, Western
blot analysis was performed. Splenocytes from wild-type
(+/+), heterozygous (+/ki), and homozygous (ki/ki) animals were analyzed. No differences in the steady-state levels
of p105, p100, p50, and RelA were found between wild-type
and knock-in splenocyte extracts (Fig. 2 A). As expected,
I
B
protein was absent in the homozygous ki/ki animals, whereas the T7-Tag antibody detected a protein that migrated to the identical position as the I
B
protein in +/ki
and ki/ki splenocyte extracts. Consistent with the presence of
extra I
B
molecules in the heterozygous and homozygous
animals, an increase was observed in the level of I
B
protein
in these animals compared with wild-type animals.
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To demonstrate that the introduced IB
could complex
with NF-
B subunits, coimmunoprecipitations were performed (Fig. 2 B). Wild-type and knock-in thymocytes were
labeled with [35S]methionine and immunoprecipitated with
antiserum against RelA and cRel under native conditions.
The immunoprecipitates were denatured and then sequentially immunoprecipitated with I
B
, T7-Tag, and I
B
antiserum. Fig. 2 B shows that in wild-type cells a large
amount of I
B
was associated with RelA and/or cRel,
whereas only a small amount of I
B
was associated with
these subunits. On the other hand, in homozygous knock-in
cells, a large amount of T7-Tag-I
B
was associated with
RelA and/or cRel. The amount of T7-Tag-I
B
in homozygous cells was comparable to that of I
B
in wild-type
cells. This indicates that the I
B
molecule introduced by
homologous recombination was expressed at a similar level
to I
B
in the wild-type animals, and that the introduced
I
B
is capable of interacting with the NF-
B subunits.
In IB
-deficient animals, a prominent increase in the
basal level of NF-
B activity was observed in spleen and
thymus, emphasizing the importance of I
B
in controlling the basal NF-
B activity in hematopoietic organs (5, 6,
18). Thus, we determined the basal levels of NF-
B activity in thymus, spleen, brain, and embryonic fibroblasts from
control and mutant mice using EMSA. Basal NF-
B activity in ki/ki animals remained relatively similar to that in the
wild-type animals in all cell types examined (Fig. 2 C), indicating that the introduced I
B
has replaced the role of
I
B
in controlling basal levels of NF-
B activity. Furthermore, NF-
B activity in nonhematopoietic cells was not
significantly altered, indicating that the presence of extra
I
B
molecules did not affect the basal machinery controlling NF-
B in these cells.
A significant feature that distinguishes IB
and I
B
is the inducibility of I
B
expression by NF-
B. After stimulation with NF-
B inducers, NF-
B accumulates in the nucleus
and stimulates ikba transcription. Transcriptional stimulation of the I
B
gene is mediated by several
B enhancer
elements present in its 5' flanking region (19, 20) and leads
to rapid accumulation of I
B
molecules, which in turn
inhibit NF-
B response. It has been proposed that this regulatory loop is responsible for the transient induction of
NF-
B activity. In contrast, the 5' upstream region of the
I
B
gene does not contain functional
B enhancers, and its transcription is not regulated by NF-
B (11). This fundamental difference in regulation might be part of the functional differences that exist between I
B
and I
B
. Since
the I
B
gene introduced by homologous recombination
was placed under the control of the ikba promoter, it
should become inducible by NF-
B. To test this inducibility and assess the functional consequence of this induction, we stimulated thymocytes from wild-type and ki/ki animals with PMA and PHA and analyzed NF-
B activity by
EMSA (Fig. 3 A). Similar to the wild-type thymocytes, ki/ki
thymocytes exhibited a rapid increase in NF-
B activity 15 min after PMA/PHA stimulation. The increase persisted
for 6 h in wild-type and ki/ki cells, indicating that the
knock-in thymocytes were capable of eliciting signal-dependent NF-
B response. Western blot analysis revealed
that, similar to I
B
in the wild-type, the T7-Tag-I
B
in
ki/ki thymocytes reaccumulated 60 min after stimulation.
The time course of reappearance for T7-Tag-I
B
is similar
to that for I
B
in the wild-type, indicating that the I
B
gene placed downstream of the I
B
promoter is inducible by NF-
B (Fig. 3 B). However, in spite of the increased
level of I
B
protein in basal condition and after induction,
signal-dependent NF-
B response in the knock-in thymocytes remained relatively similar to the wild-type.
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A tissue-specific difference in function has been
proposed between IB
and I
B
. Based on the relative
abundance of these two proteins, and on the fact that disruption of I
B
specifically affected NF-
B activity in hematopoietic cells, it has been postulated that I
B
plays a
more important role in hematopoietic cells, whereas I
B
is more important in nonhematopoietic cells (18). To assess
the functional consequence of the replacement in nonhematopoietic cells, we tested NF-
B response in fibroblasts
from 15-d-old embryos (Fig. 4). Wild-type and ki/ki primary mouse embryo fibroblasts (MEFs) were treated with
TNF-
for various periods of time and harvested for
EMSA and Western blot analysis. After 30 min of TNF-
treatment, NF-
B was activated in both ki/ki and wild-type MEFs (Fig. 4 A). The level of NF-
B induction was
similar, indicating that the knock-in fibroblasts are capable
of eliciting a normal NF-
B response. However, NF-
B
activity in ki/ki cells diminished to near basal level 3 h after
stimulation, whereas NF-
B activity in the wild-type remained prominent at the same time point. A Western blot
analysis showed that the T7-Tag-I
B
in ki/ki cells was
induced after TNF-
stimulation, resulting in accumulation of a significantly higher amount of I
B
in the knock-in cells than in the wild-type cells (Fig. 4 B). The time at
which the T7-Tag-I
B
accumulates dramatically in the
cell correlates to the time point at which NF-
B activity
becomes noticeably lower in the ki/ki fibroblasts.
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Studies using primary fibroblasts from IB
-deficient embryos have
demonstrated the importance of I
B
in postinduction repression. NF-
B activity in fibroblasts lacking I
B
persisted for >2 h after TNF-
removal, whereas NF-
B activity in wild-type fibroblasts quickly returned to basal level
(within 30 min after stimulation; references 5, 6). To determine if the I
B
introduced by homologous recombination was capable of inhibiting the NF-
B activity after induction, we performed postinduction experiments using the ki/ki fibroblasts (Fig. 5). Wild-type and ki/ki primary
embryo fibroblasts were treated for 30 min with TNF-
,
after which TNF-
was removed, and the cells were harvested 60, 120, and 240 min later. In the wild-type MEFs,
NF-
B activity returned to basal levels nearly 60 min after
TNF-
removal (Fig. 5 A). In ki/ki MEFs, the return of
NF-
B to basal levels occurs on or after 120 min. A Western blot analysis revealed that, similar to I
B
, the T7-Tag-I
B
was upregulated after induction. However, the
time at which maximal T7-Tag-I
B
accumulated was
delayed compared to I
B
(60 min versus 2 h; Fig. 5 B).
Peak T7-Tag-I
B
accumulation corresponded to the
time at which NF-
B activity diminished to basal level.
Therefore, in the context of the I
B
promoter, I
B
can
be induced by NF-
B and is competent in postinduction
repression, although the time course of repression is slower
than that of I
B
in the wild-type cells.
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![]() |
Discussion |
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Using the knock-in approach, we have generated mice
that carry a replaced IB
gene. A T7-Tag-modified I
B
gene was expressed under the promoter and the 5' regulatory sequence of the I
B
gene. As a result, the ikba allele
was inactivated, and the introduced ikbb was expressed and
regulated like ikba. This approach allowed us to investigate
the functional similarity of I
B
and I
B
under physiological conditions. The knock-in mice survived to adulthood and did not display any of the abnormalities that are
present in the I
B
-deficient mice. Basal levels of NF-
B
activity in the hematopoietic organs of the knock-in mice
were not elevated like those in the I
B
-deficient mice.
Furthermore, signal-induced NF-
B response in thymocytes was also normal. These results indicate that I
B
can functionally compensate for the absence of I
B
.
The notion that IB
and I
B
are biochemically
equivalent is unexpected. Although many similarities exist
between these two molecules, significant biochemical differences have also been reported. For example, I
B
degradation is induced by a wide variety of NF-
B inducers,
but only a subset of these inducers effects I
B
degradation
(11, 21). The kinetics of degradation is also different between the two molecules. In general, I
B
has a more
rapid degradation than I
B
. Differences in the basal phosphorylation state, degradation mechanism (21), and affinity
to RelA (22) have also been shown for these two molecules. These observations formed the basis of hypotheses
that the biochemical differences of I
B
and I
B
contribute to functional differences for these two molecules.
One prominent functional difference that has been proposed for the IBs is the unique "chaperone" role of I
B
(23). I
B
molecules resynthesized after stimulation have
been found to be hypophosphorylated. The hypophosphorylated I
B
molecules can bind to NF-
B complexes, but
do not mask the nuclear localization signals of NF-
B.
Thus, they serve as chaperones for NF-
B complexes, protecting them from inhibition by the resynthesized I
B
molecules and, therefore, maintaining the persistent activation of NF-
B. However, recent reports have provided evidence against this model. For example, Miyamoto et al.
have reported that inhibition of I
B
degradation by proteasome inhibitors does not have any effect on the constitutive activity of NF-
B in WEHI cells (21). Prolonged nuclear localization of NF-
B complexes also is not necessarily associated with long-term depletion of I
B
(24).
Moreover, mice deficient in I
B
have been also generated (Attar, R.M., manuscript in preparation). NF-
B responses elicited by various extracellular stimuli, along with
various immune responses in these mice, are indistinguishable from those in wild-type mice, suggesting that persistent NF-
B activation can take place in the absence of
I
B
. Furthermore, NF-
B activity in thymocytes remained relatively unchanged in the knock-in mice, indicating that the resynthesized I
B
s did not chaperone
more NF-
B complexes into the nucleus, although a large
excess of T7-Tag-I
B
is produced after stimulation (Fig.
3). It is surprising that such a large amount of I
B
did not
affect NF-
B activity. It appears as if the NF-
B complexes in the knock-in cells are protected from the resynthesized T7-Tag-I
B
just as the NF-
B complexes in the
wild-type cells are protected from the resynthesized I
B
.
Possibly, a separate mechanism exists that is independent of
the phosphorylation status of I
B
for maintaining the persistent activation of NF-
B.
In light of the fact that the mice lacking IB
but expressing I
B
controlled by the ikba promoter regulatory
elements appear healthy, we conclude that the two I
B
molecules are biochemically equivalent, and that certain
biochemical features reported on I
B
, such as hypophosphorylation or slow degradation, do not contribute significantly to the functional difference between them. We postulate that the difference in relative level of expression between these two molecules is a primary component of
their functional difference. It has been reported that these
two I
B molecules are expressed differently in different tissues: I
B
mRNA is highly abundant in the spleen,
whereas I
B
mRNA is mostly abundant in the testis (11).
I
B
protein expression is also known to be higher in the
hematopoietic organs, whereas I
B
protein is distributed equally between the hematopoietic and nonhematopoietic
tissues (5). Consistent with these observations, we are able
to immunoprecipitate a much larger amount of I
B
than
I
B
molecules in our thymocyte preparation (Fig. 2 B
and data not shown). Additionally, our Western blots also
show that the I
B
introduced by homologous recombination is expressed at a higher level than the endogenous
I
B
molecules in thymocytes (Fig. 3 B). On the other
hand, the relative increase in I
B
level does not appear to
be as high in the knock-in fibroblasts (Fig. 4 B). Although
we can not rule out that the differences in stability may
have contributed to some of the difference in steady state
expression level, our observations suggest that the ikba promoter is more active in the hematopoietic system compared with nonhematopoietic systems.
In addition to tissue-specific differences in expression,
the autoregulatory feature is also a fundamental difference
between the two molecules. It has been proposed that
NF-B-inducible regulation of I
B
gene transcription is
important for terminating NF-
B response in nonhematopoietic cells. By performing a postinduction repression experiment similar to that performed in the study of I
B
-deficient mice, we found that the T7-Tag-I
B
molecule
can serve as an inhibitor similar to I
B
in postinduction
repression. This again supports the notion that I
B
and
I
B
are biochemically equivalent, and that regulatory
B
enhancer elements upstream of the genes are crucial components of the functional differences between these I
Bs. A
more recently identified member of the I
B family, I
B
,
also has this autoregulatory feature (25, 26). The fact that
some, but not all, members of the I
B family share this
property further implicates the importance of specific regulatory sequences in conferring different functions of these
molecules. This auto-regulatory feature appears to be closely
linked to tissue-specific difference in expression, as resynthesis of excess T7-Tag-I
B
is associated with a different
effect in fibroblasts than in thymocytes.
Despite the increase in understanding NF-B function,
the specificity and physiological relevance of the different
Rel/NF-
B and I
B proteins remains unclear. The knockouts and transgenic animals have provided important information on the physiological roles of the individual members (7, 27), but functional redundancy probably has
masked the full importance of each member. Besides knowledge provided from single and double knockout
studies, the knock-in approach provides a powerful tool for
analysis of redundancy and the physiological function of individual members. The knock-in approach could be used
to dissect the importance of specific domains or structures
within a molecule in conferring specificity. Similar to this
study, the knock-in approach has been used to reveal redundancy of two other transcription factor families, the
Engrailed family (En-1 and En-2; reference 36), and the
MyoD family (Myogenin and Myf-5; reference 37). In
both cases, replacement of a similar member of the same
family resulted in complete rescue of the phenotype from
single knockout. Together, these data demonstrate that
many closely related members of a gene family acquire different functions in evolution through divergence of gene
expression, rather than through divergence in biochemical
function. Because overlapping gene function is likely to be
prevalent in mammals, such approaches are critical for clarifying the unique and overlapping function of members of
a family, and for determining the complete repertoire of
functions of individual genes.
![]() |
Footnotes |
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Address correspondence to Rodrigo Bravo, Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Company, PO Box 4000, Princeton, NJ 08543-4000. Phone: 609-252-5744; Fax: 609-252-6051; E-mail: bravo#m#_rodrigo.prilvms1{at}msmail.bms.com
Received for publication 9 April 1998 and in revised form 8 July 1998.
We thank Chery Rizzo for cell culture assistance, Mavis Swerdel and Alice Lee for chimera aggregations, Kenneth Class for flow cytometry, and the staff of Veterinary Sciences at Bristol-Myers Squibb for their excellent support. We also thank Donald Hawken for support and critical reading of the manuscript.
Abbreviations used in this paper
EMSA, electrophoretic mobility shift assay;
ES, embryonic stem;
MEF, mouse embryo fibroblast, NF-B, nuclear
factor
B.
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