Diabetes Unit and Medical Services (C.B., N.N., M-A.B.)
Massachusetts General Hospital Harvard Medical School Boston,
Massachusetts 02114
Department of Medical Nutrition (A.M.,
P.T., J-Å.G.) Karolinska Institute Novum, S-14186 Huddinge,
Sweden
National Research Council (H-F.Z.) Biotechnology
Research Institute Montreal, Quebec H4P 2R2, Canada
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ABSTRACT |
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INTRODUCTION |
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The cytochrome P450 2C12 gene (CYP2C12) encodes a
steroid sulfate 15ß hydroxylase that is induced in the liver of
female rats during puberty (10, 11). In contrast, the cytochrome
P450 2C11 (CYP2C11) gene encodes a steroid 16- and
2
-hydroxylase that is induced in the liver of male rats during
puberty (12, 13, 14). Differential expression of these genes in male and
female rats is set by sexually dimorphic patterns of GH secretion and
is mediated at the level of transcription initiation (11, 13, 15). In
male rats, GH is secreted in episodic bursts every 3 to 4 h with
low or undetectable levels in between. GH pulses of lower amplitudes
occur more frequently in females, and base line levels are higher than
in males, resulting in a continuous pattern of GH secretion (16, 17).
The male pattern of GH secretion activates the transcription factor STAT5b (18, 19). Furthermore, in male mice disruption of the STAT5b gene results in loss of male-specific liver CYP gene expression (20). Thus, it is presumed that STAT5b regulates CYP2C11 gene expression in rat liver (18, 19). Both the male and female pattern of GH secretion can induce expression of hepatic nuclear factor-6 (HNF-6), but the female pattern is somewhat more effective. HNF-6 can bind and enhance transcription from the CYP2C12 promoter (21).
The female pattern of GH secretion leads to the marked induction of an
unidentified liver nuclear protein complex referred to as the
GH-regulated nuclear factor (GHNF) (22). In liver extracts isolated
from female rats, GHNF binds several regions in the CYP2C12
promoter and at least one region in the CYP2C11 promoter.
One of the GHNF-binding sites in the CYP2C12 gene (-231 to
-185) contains a putative binding site for the CCAAT/enhancer-binding
protein (C/EBP
).
C/EBP is an important determinant of liver gene expression (23, 24, 25, 26, 27, 28, 29, 30)
and can activate the expression of both the CYP2C12 and
CYP2C11 genes (14, 31). In primary hepatocytes, we have
shown that C/EBP
stimulates expression of the CYP2C12
gene through the sequence, 5'-TTATCAATGTT (-229 to -207) (31).
However, regulation of C/EBP
alone does not appear to account for
female-specific expression of the CYP2C12 gene (22, 31).
We noticed that the C/EBP site in the CYP2C12 gene
carries a recognition sequence for SRY-like HMG proteins,
5'-PyCA/TTTGA/TA/T,
on the antisense strand (2, 32, 33, 34). In this report we examined whether
IRE-ABP could modulate the effect of C/EBP
on expression of the
CYP2C12 gene expression. We show that, in primary
hepatocytes, overexpression of IRE-ABP inhibits C/EBP
-mediated
activation of the CYP2C12 gene, but not the control gene
CYP2C11. This observation correlates with the presence of
overlapping binding sites for C/EBP
and IRE-ABP in the
CYP2C12 promoter, but not the CYP2C11 promoter.
We propose that the overlap in DNA-binding specificity between IRE-ABP
and activators, such as C/EBP
in certain target genes, is one
mechanism by which SRY-like proteins may modulate the expression of
sex-specific genes.
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RESULTS |
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We next examined whether IRE-ABP and C/EBP together could regulate
the expression of chloramphenicol acetyl transferase (CAT) reporter
genes driven by the CYP2C12 and CYP2C11 promoters
(Fig. 2
). In transiently transfected
NIH-3T3 cells, IRE-ABP had no effect on basal expression of the
CYP2C12- and CYP2C11-CAT constructs (Fig. 2A
). In
the absence of IRE-ABP, C/EBP
stimulated CYP2C12- and
CYP2C11-CAT activities 11- and 24-fold, respectively (Fig. 2A
). In the presence of IRE-ABP, however, C/EBP
-stimulated
CYP2C12-CAT activity was inhibited 80% (Fig. 2A
, left), while IRE-ABP had no effect on C/EBP
-stimulated
CYP2C11-CAT activity (Fig. 2A
, right).
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IRE-ABP and C/EBP Bind to Overlapping Sites in the Promoter of
the CYP2C12, but Not the CYP2C11 Gene
To determine why IRE-ABP inhibits C/EBP-mediated activation of
CYP2C12 gene expression, but not CYP2C11 gene
expression, we used deoxyribonuclease I (DNase I) footprint analysis to
identify the IRE-ABP- and C/EBP
-binding sites in these genes.
C/EBP
and IRE-ABP were expressed as
glutathione-S-transferase (GST) fusion proteins, affinity
purified on glutathione Sepharose beads, and eluted with glutathione or
cleaved with thrombin to remove GST.
As shown in Fig. 3A, GST-C/EBP
(0.150.9 µg) bound to two regions in the CYP2C12
promoter, as compared with GST alone (Fig. 3A
, compare lanes 25 to
lane 1; see stippled circles). Thrombin cleaved IRE-ABP
(Fig. 3A
, lanes 710), and GST-IRE-ABP (Fig. 3A
, lanes 1112) bound
to four regions in the CYP2C12 promoter of which only
IRE-ABP sites 24 are shown (Fig. 3A
, compare lanes 10 and 12 to lane
6). While IRE-ABP binding to sites 2 and 3 is clearly demonstrated by
DNase I footprinting, binding to site 4 was better demonstrated by
electrophoretic mobility shift assay (EMSA) (see Fig. 4A
, lane 1). In
the CYP2C12 promoter, the two C/EBP
-binding sites overlap
with IRE-ABP binding sites 2 and 4. In contrast, in the
CYP2C11 promoter, GST-C/EBP
(0.060.6 µg) bound to
three sites (Fig. 3B
, compare lanes 1 and 2 to lanes 35; see
stippled circles), each of which was distinct from the one
region bound by GST-IRE-ABP (0.060.6 µg), referred to as Site A
(compare lanes 1 and 2 to lanes 68; see solid
rectangles).
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In the CYP2C11 gene promoter, there is one IRE-ABP-binding
site located between nt -113 to -101, referred to as site A (see Fig. 3C). Site A contains the sequence 5'-TTCTTTGTG (see Table 1
) and does
not bind C/EBP
. Three C/EBP
-binding sites were detected in the
CYP2C11 promoter; two upstream (-194 to -153 and -148 to
-132) and one downstream (-88 to -69) of IRE-ABP-binding site A
(Table 2
). These C/EBP
sites do not carry an HMG consensus sequence
and do not bind IRE-ABP (Fig. 3B
, lanes 68). Therefore, although both
IRE-ABP and C/EBP
bind the 5'-flanking sequences of the
CYP2C11 gene, their binding sites do not overlap.
A schematic diagram depicting the location of the binding sites for
IRE-ABP and C/EBP in the CYP2C12 and CYP2C11
promoters and their relationship to previously described sex-specific
and GH-dependent sites is shown in Fig. 3C
. IRE-ABP-binding site 4
in the CYP2C12 promoter as well as IRE-ABP-binding site A in
the CYP2C11 promoter overlap sequences bound by the
GH-regulated nuclear factor GHNF, previously described by Waxman
et al. (22). Sex-specific and GH-dependent DNase I
hypersensitivity sites have also been identified in the
CYP2C11 and CYP2C12 promoters using liver nuclear
extracts from male and female rats (14, 15, 22). In the
CYP2C12 gene, the sex-specific and GH-responsive DNase I
hypersensitivity sites are located at nt -99 and -140, respectively
(15). A female-specific hypersensitive region also exists between nts
-112 and -159 (15). C/EBP
and IRE-ABP cause hypersensitivity to
DNase I in these regions. In particular, binding of IRE-ABP to
CYP2C12 increased sensitivity to DNase I near nt -120,
which is part of binding site 2 (Fig. 3A
, lanes 712).
IRE-ABP Is a Component of the Liver Nuclear Protein Complexes That
Bind CYP2C11 and CYP2C12 DNA
We have previously reported that C/EBP is a component of the
rat liver protein complex that binds site 4 in the CYP2C12
DNA (31). To determine whether IRE-ABP also binds site 4, we used
specific antibodies raised against IRE-ABP and C/EBP
to detect these
proteins in EMSA gels (Fig. 4A
). The results shown below were obtained
in three independent liver nuclear extracts isolated from male rats in
which we expect the female CYP2C12 gene to be repressed.
Under the conditions of this assay, no significant difference in
binding pattern was seen in extracts prepared from male and female rats
(data not shown).
Purified IRE-ABP protein was preincubated with partially purified
preimmune (PI) chicken IgY (Fig. 4A, lane 1) or immune (I) chicken IgY
raised against the full-length IRE-ABP protein (Fig. 4A
, lane 2) before
addition of CYP2C12 site 4 DNA. In the presence of the
IRE-ABP antibody, purified IRE-ABP was completely supershifted (Fig. 4A
, compare lane 1 to lane 2).
Next we examined the binding of liver nuclear extract to wild-type site
4 DNA and a mutant of site 4 with the wild-type HMG consensus sequence
5'-ACAATGT changed to 5'-CGCCCTG (Fig. 4A, lanes 36, as compared with
lanes 79). Several complexes were detected with wild-type site 4 DNA
(Fig. 4A
, lane 3, complexes I-V). In the presence of IRE-ABP antibody,
two of the complexes (I and II) were diminished in intensity (Fig. 4A
, lane 4) as compared with the PI control (Fig. 4A
, lane 3), and two new
supershifted (ss) bands appeared (Fig. 4A
, lane 4). Complex I
comigrates with bacterial IRE-ABP. Thus, the IRE-ABP antibodies shift
bacterial IRE-ABP as well as an activity that comigrates with IRE-ABP
in liver nuclear extracts (complex I on the CYP2C12 probe). In the
presence of antibody to IRE-ABP, the intensity of complexes II, IV, and
V was diminished as well. Thus, there are IRE-ABP-like activities that
migrate with lower mobility than pure IRE-ABP in liver nuclear extract.
These differences in migration may be the result of posttranslational
modifications, interaction of IRE-ABP with other proteins such as
C/EBP
, or binding of other IRE-ABP-like gene products.
When the binding was performed using the mutant probe, complex I was
not detected (compare lanes 3 and 7). The mutant probe also failed to
show the two supershifted IRE-ABP bands (Fig. 4A, lane 8). Furthermore,
although the mutant probe bound to new complexes that migrated
similarly to complexes IIV, these complexes were markedly less
sensitive to the IRE-ABP antibody (Fig. 4A
, compare lanes 3 and 4 to
lanes 7 and 8). Thus, IRE-ABP antibodies detect an IRE-ABP-like binding
activity in the complex that binds site 4.
The EMSA gel conditions used to detect IRE-ABP do not favor
binding of C/EBP complexes. Nevertheless, preincubation of a
C/EBP
antibody with liver nuclear extract supershifted a minor band,
complex III, that bound to the wild-type but not the mutant site 4 DNA
probe (Fig. 4A
, compare lanes 3 and 5 to lanes 7 and 9). Therefore,
although the mutant probe detects a band that comigrates with complex
III, the new complex does not contain C/EBP
.The supershift complex
was more complicated in the presence of both the C/EBP
and IRE-ABP
antibodies than with either antibody alone, but multiple attempts
failed to resolve these complexes (Fig. 4A
, lane 6).
To confirm that IRE-ABP-binding site A in the CYP2C11 gene
promoter binds IRE-ABP but not C/EBP, we examined the binding of
purified IRE-ABP and C/EBP
proteins to this site (Fig. 4B
, lanes 1
and 2, respectively). As expected from the DNase I protection assay,
IRE-ABP bound site A but C/EBP
did not. Several liver nuclear
complexes were detected with wild-type site A DNA (Fig. 4B
, lanes
36). Preincubation of male liver nuclear extract with an antibody to
IRE-ABP led to a marked decrease in intensity of a prominent complex
(Fig. 4B
, lane 4), as compared with preincubation of this extract with
PI serum (lane 3). We presume that the complex was supershifted and
that the complex did not enter the well (Fig. 4A
, lane 4). When
compared with nonimmune (NI) IgG (Fig. 4B
, lane 5) a specific antibody
to C/EBP
(IgG) did not alter binding of liver nuclear complexes to
site A (Fig. 4B
, lane 6). The major IRE-ABP-like activity detected with
the CYPC11 probe (see Fig. 4B
, lane 4) migrates similarly to
an IRE-ABP activity (complex II) detected with the CYP2C12
probe (see Fig. 4A
, lane 4). While the CYP2C11 site A probe
detects only one major band, the CYP2C12 site 4 probe
detects several bands that are additional bands that are inhibited by
the IRE-ABP antibody. We suspect these additional complexes represent
IRE-ABP in complex with other proteins (such as C/EBP
).
Combined, these observations demonstrate that the CYP2C12
site 4 binds both C/EBP and IRE-ABP (or an IRE-ABP-like activity),
while the CYP2C11 site A binds IRE-ABP and not C/EBP
.
IRE-ABP and C/EBP Modulate Gene Expression via
Overlapping Target Sites Examined on a Heterologous Promoter
The functional effect of C/EBP on the CYP2C12 gene
maps to sequences located between nts -220 and -207 (31). Having
shown that both IRE-ABP and C/EBP
are components of the nuclear
protein complex that binds site 4 in the CYP2C12 promoter,
we examined whether the inhibitory effect of IRE-ABP on C/EBP
could
be mapped to this region. The IRE-ABP binding sites at site 4 extend
upstream and downstream of the C/EBP
footprint, from nts -231 to
-184. To capture the full effect of IRE-ABP, we cloned nts -231 to
-184 upstream of a heterologous promoter. Having shown that IRE-ABP
does not inhibit C/EBP
-stimulated CYP2C11-CAT activity
(Fig. 2
), we chose to use nt -65 to +26 of this promoter, referred to
as the minimal CYP2C11 promoter (min-CAT), to drive
expression of the CAT reporter gene (site 4·min-CAT).
In CHO cells, C/EBP alone did not stimulate min-CAT activity.
Although IRE-ABP alone inhibited the basal activity of min-CAT by 50%,
there was no effect of IRE-ABP alone on basal expression of the site
4·min-CAT construct (Fig. 5
, panels A
and B). In the absence of IRE-ABP, C/EBP
stimulated site 4·min-CAT
activity 4-fold, whereas in the presence of IRE-ABP, the ability of
C/EBP
to stimulate site 4·min-CAT activity was completely blocked
(Fig. 5B
). Therefore, sequences located between nt -231 to -183 in
the CYP2C12 gene promoter confer activation of gene
transcription by C/EBP
and inhibition of this effect by IRE-ABP.
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We engineered two point mutations in the HMG domain of IRE-ABP, one of which inhibits and the other of which enhances binding to DNA in the setting of the human SRY protein (C. Buggs and M. Alexander-Bridges, unpublished observations). In the binding- deficient mutant, a glycine is substituted for tryptophan at position 12 in the HMG box (IRE-ABP·Gly), an invariant residue among all HMG box proteins; whereas in the binding-enhanced mutant, a threonine is substituted for proline at position 25 (IRE-ABP·Thr), a substitution that is tolerated in certain HMG-box proteins (40) such as LEF-1 (41). In the setting of SRY, both mutations are associated with sex reversal in humans.
Wild-type IRE-ABP alone (Fig. 7, lane 1),
when added at concentrations one third of those used in Fig. 6
, forms
two complexes on site 4 DNA, with the monomer as the major complex. In
contrast, the larger quantity of IRE-ABP added in Fig. 6
resulted in
the predominant appearance of the IRE-ABP dimer complex (Fig. 6
, lane
1). The IRE-ABP·Gly mutant does not bind to site 4 DNA (Fig. 7
, lane
4), whereas the IRE-ABP·Thr mutant shows enhanced binding to site 4;
in particular, more of the dimer form is recovered (Fig. 7
, lane
6).
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To determine whether the inhibitory effect of IRE-ABP might be
mediated, in part, by an interaction with C/EBP, we used an in
vitro protein interaction assay (42). In vitro
translated C/EBP
was incubated with equivalent amounts of GST or
GST·IRE-ABP bound to Sepharose beads. The beads were washed and the
eluate subjected to electrophoresis as described in Materials and
Methods. In vitro translated C/EBP
bound to
GST·IRE-ABP, but not to GST alone (Fig. 8
, compare lane 3 to lane 2).
Furthermore, C/EBP
interacted with the HMG DNA-binding domain of
IRE-ABP (data not shown), which led us to determine whether mutations
within the HMG domain might affect the ability of IRE-ABP to interact
with C/EBP
as well as to bind DNA. In comparison to wild-type
GST/IRE-ABP, which bound more than 25% of the input C/EBP
(Fig. 8
, lane 3 as compared with lane 1), the GST/IRE-ABP·Gly mutant bound
very little C/EBP
(Fig. 8
, lane 4). In contrast, the
GST/IRE-ABP·Thr mutant bound amounts comparable to wild-type IRE-ABP
(Fig. 8
, lane 5 as compared with lane 3). Therefore, a single point
mutation in the HMG domain of IRE-ABP blocks its ability to bind DNA
and interact with C/EBP
in vitro. Ethidium bromide
intercalates DNA and inhibits the interaction of DNA with proteins. In
the presence of ethidium bromide (200 µg/ml), the wild-type and
mutant IRE-ABP proteins bind C/EBP
to a comparable extent (lanes
79). Thus, we conclude that the direct interaction of C/EBP
and
IRE-ABP is not affected by mutations in the HMG-binding domain of
IRE-ABP.
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DISCUSSION |
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Previous reports suggest that the ability of SRY-like proteins to
activate transcription may be a prominent feature of this family of
genes (7, 8, 9, 43, 44). For example, SRY and related proteins, such as
SOX4, have been shown to bind and activate transcription through
multimerized 5'-ACAAT sequences driving a heterologous promoter (7, 8).
In the setting of a native promoter, however, we find that IRE-ABP
binds the sequence 5'-ACAAT in the CYP2C12 gene and acts as
a context-specific repressor of C/EBP-activated gene transcription.
IRE-ABP inhibited C/EBP
-stimulated CYP2C12 expression in
three separate assay formats, using: 1) the endogenous
CYP2C12 gene (Fig. 1
), 2) the native CYP2C12
promoter to drive expression of a CAT reporter gene (Fig. 2
), and 3)
the monomeric site 4 from the 5'-flanking region of the
CYP2C12 gene to drive expression of a heterologous promoter
(Fig. 5
). In contrast, IRE-ABP did not footprint the C/EBP
sites
that mediate activation of the CYP2C11 gene by C/EBP
, nor
did C/EBP
footprint the IRE-ABP site in the CYP2C11 gene
(see Fig. 3C
). Thus, the male-specific CYP2C11 gene, wherein
the IRE-ABP site does not overlap with C/EBP
sites, serves as a
control gene whose expression can be examined in the setting of a
native promoter. The observation that IRE-ABP has no effect on C/EBP
activation of CYP2C11 suggests that the ability of IRE-ABP
to inhibit C/EBP
-activated transcription of CYP2C12
requires that the two proteins bind to adjacent or overlapping DNA
sequences. These findings provide new insights into the molecular
mechanisms that underlie the ability of SRY-like architectural proteins
to regulate gene transcription during differentiation and in response
to hormonal stimuli.
C/EBP and IRE-ABP can interact directly, independent of their
ability to bind DNA. Nevertheless, several observations indicate that
the ability of IRE-ABP to inhibit C/EBP
-activated CYP2C12
gene expression depends primarily on binding of IRE-ABP to
CYP2C12 DNA rather than to C/EBP
. The direct
protein-protein interaction between IRE-ABP and C/EBP
can be
visualized in the presence of ethidium, which intercalates DNA and
prevents DNA-dependent protein interactions. Under these conditions,
the interaction of C/EBP
with IRE-ABP is equivalent for wild-type
IRE-ABP, the IRE-ABP·Gly mutant that is deficient in DNA binding, and
the IRE-ABP·Thr mutant that enhances binding to DNA (Fig. 8
, lanes
68). Thus, the IRE-ABP·Gly mutant that inhibits DNA-dependent
protein interactions between IRE-ABP and C/EBP
does not inhibit the
direct interaction of these proteins (Fig. 8
, compare lanes 3 and 4).
Nevertheless, the IRE-ABP·Gly mutant lacks the ability to inhibit
C/EBP
-activated CYP2C12 gene expression. Moreover, the
C/EBP
- activated expression of CYP2C11 is not inhibited
by wild-type IRE-ABP, indicating that the protein-protein interaction
is not sufficient to cause inhibition of C/EBP
action at this gene.
Thus, although we cannot conclusively distinguish whether the ability
of IRE-ABP to inhibit C/EBP
action on the CYP2C12 gene
results from simple competition for the activator site or from a
protein-protein interaction that occurs once these proteins are bound
to adjacent sites on DNA, our data indicate that repression of C/EBP
by IRE-ABP requires IRE-ABP binding to DNA.
We propose that once bound to DNA, IRE-ABP modulates C/EBP-mediated
transcriptional activation via one or more mechanisms: 1) IRE-ABP could
simply displace C/EBP
from DNA, 2) IRE-ABP could interact with
C/EBP
so as to mask the interaction of C/EBP
with the
transcriptional machinery, or 3) IRE-ABP could bend CYP2C12
DNA so as to prevent C/EBP
from interacting with components of the
transcriptional machinery. In support of the third mechanism,
sequence-specific HMG box proteins are thought to activate
transcription by bending DNA and facilitating the assembly of
nucleoprotein complexes (40, 45, 46, 47). For example, studies indicate
that the T cell-specific HMG box protein LEF-1 stimulates gene
expression by binding and bending DNA, which, in turn, facilitates
interactions between transcription factors bound to sequences flanking
the HMG site (41). Inasmuch as similar results have been obtained with
SRY (41), it seems plausible that IRE-ABP could inhibit activation of
CYP2C12 gene expression by inducing a bend in DNA that
inhibits interactions between C/EBP
and neighboring proteins.
Although this mechanism does not require that IRE-ABP interact with
C/EBP
, it does not exclude a contribution from such a direct
interaction.
In this study we found that IRE-ABP can differentially modulate the expression of two sexually dimorphic cytochrome P450 genes, CYP2C11 and CYP2C12, which are differentially regulated at the level of transcription initiation in the liver of adult male and female rats (11, 13, 15). This regulation in rodents has been shown to be attributable to a sexually dimorphic pattern of GH secretion: the male rat secretes GH in a pulsatile pattern, whereas the female rat secretes GH in a more continuous pattern (16, 17). These two patterns of GH secretion cause differential activation/induction of transcription factors. The transcription factor STAT5b is activated by pulsatile GH secretion (18, 19) and activates expression of male-specific CYP genes in mice (20). In contrast, the HNF-6 transcription factor, whose expression is dependent on GH, is more efficiently induced by the relatively continuous pattern of GH secretion that occurs in female rats, and HNF-6 can increase transcription from the female CYP2C12 promoter (21).
In addition to HNF-6, continuous GH secretion induces binding of an as yet unidentified protein complex, GHNF, which binds to sequences in both the CYP2C12 and the CYP2C11 promoters (21, 22). Thus, three or more transcription factors (STAT5b, HNF-6, and GHNF) have been implicated as mediators of the differential effect of GH signaling on the expression of the CYP2C12 and CYP2C11 genes.
In this study we show that IRE-ABP binds to two distinct motifs,
5'-TCAATG/AT/A in the
CYP2C12 gene and 5'-TCTTTGT in the CYP2C11 gene,
both of which conform to the consensus sequence for SRY-like proteins,
5'-PyCA/TTTGA/TA/T.
In the CYP2C12 gene, IRE-ABP binds to four sites, most
tightly to sites 2 and 4. Site 1 overlaps the HNF-6 site,
5'-AAATCAATAT, located between nts -38 to -47 (see Table 1) (21). As
shown in Fig. 3C
, IRE-ABP binding Site 4 (-231 to -183), which is
also bound by C/EBP
(-222 to -204) overlaps a GHNF-binding site
(-231 to -185) (22). In the CYP2C11 gene, the single
high-affinity IRE-ABP binding site A (-115 to -105) also overlaps the
GHNF binding site (-135 to -85) (see Fig. 3C
), but this site,
5'-TCTTTGT, is not a target for C/EBP
(22). Thus, the binding of
IRE-ABP overlaps with that of GHNF in both the CYP2C12 and
CYP2C11 genes, despite the fact that the structure of the
binding sites is different in each gene. Therefore, IRE-ABP (or an HMG
protein with similar specificity) may modify the binding or activity of
the putative transactivator GHNF.
IRE-ABP is itself unlikely to be GHNF, because we did not detect the
marked female-specific differences in IRE-ABP-binding activity that
have been observed for GHNF. Furthermore we have not seen differences
in IRE-ABP mRNA levels in the liver of male and female rats (our
unpublished observations). We speculate that IRE-ABP, in addition to
its inhibition of C/EBP action on the CYP2C12 gene, also
acts as a constitutive repressor of both the CYP2C11 and
CYP2C12 genes by modifying the activity of GHNF and possibly
HNF-6.
Our results are consistent with the following model for differential
regulation of the sex-specific CYP2C12 and
CYP2C11 genes by a repressor, such as IRE-ABP in female and
male rats. Each gene contains a site (site A in CYP2C11 and
site 4 in CYP2C12) that can bind both an activator
(C/EBP) and a repressor of transcription (IRE-ABP). Depending on
which proteins are bound, expression of one gene is activated while the
other is markedly inhibited. In this study, we have used C/EBP
and
IRE-ABP to illustrate that this effect can occur; however, we can only
speculate as to how HNF-6 or GHNF may function in this context.
In male rats, expression of the male gene CYP2C11 is most
likely determined by activated STAT5b (20), which we presume overrides
an inhibitory effect of IRE-ABP on this gene. As regards expression of
the female CYP2C12 gene in the male, the relatively low
levels of the activator HNF-6 and of GHNF seen with pulsatile GH
secretion are insufficient to overcome the repressive effect of IRE-ABP
at site 4; and as seen in Figs. 1, 2
and 5, IRE-ABP can effectively
inhibit the ability of C/EBP
to activate CYP2C12 gene
expression at this site. These conditions result in the repressed state
of the female gene CYP2C12 in the male.
In the female rat, we surmise that the robust induction of GHNF
and HNF-6 overrides the negative effect of IRE-ABP and activates the
"female" CYP2C12 gene. However, CYP2C11 also
carries a GHNF-binding site. Why then is this gene repressed in the
liver of female rats and not induced by GHNF? Our data indicate that
IRE-ABP has a higher affinity for the GHNF-binding site in the
CYP2C11 gene, binding site A (see Fig. 3B), than it does for
binding site 4 in the CYP2C12 gene (Fig. 3A
). Therefore,
IRE-ABP should compete more effectively with GHNF at CYP2C11
(site A) than it does at CYP2C12 (site 4). As a result, the
male gene, CYP2C11, remains repressed in the female, despite
induction of GHNF and activation of CYP2C12.
This model does not require marked changes in expression or DNA-binding
activity of IRE-ABP, but instead relies on observed differences in
apparent affinity and specificity of IRE-ABP for its sites in the
CYP2C12 and CYP2C11 genes (Fig. 3, A and B).
However, it is clear that additional studies are required to establish
whether this model can explain the sex-differentiated expression of
CYP2C genes and to elucidate the relationship of
IRE-ABP and GHNF.
In summary, we find that IRE-ABP, an SRY-related HMG box protein, can
bind C/EBP sites that contain a CAAT box and inhibit
C/EBP
-activated gene transcription. Our findings also indicate that
SRY-like proteins can inhibit gene expression by binding to DNA so as
to interfere with the activating function of other transcription
factors; in the case of the CYP2C12 gene, C/EBP
and
IRE-ABP bind the same site. These observations illustrate a previously
unappreciated mechanism by which SRY-like proteins can modulate gene
expression during cellular differentiation and in response to hormonal
stimuli.
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MATERIALS AND METHODS |
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The CDM8·IRE-ABP expression plasmid was constructed by cloning full-length IRE-ABP cDNA into an EcoRI cut CDM8 vector (a kind gift from B. Seed, Massachusetts General Hospital). The mutant CDM8·IRE-ABP constructs were made by PCR-directed mutagenesis as follows: the reverse primer used to synthesize IRE-ABP·Gly was 5'-GCCGCTTGGAGATCTCGGCGTTGTGCATGTCGGGCGACTGCTCCATGATCTTGCGCCGCTCGATCTGCACCCCACCATAAAG-3' (the single nt substitution is underlined; the reverse primer used to synthesize IRE-ABP·Thr has the sequence 5'-GCCGCTTGGAGATCTCGGCGTTGTGCATGTCGGTCGACTGCTCCA-3' (the nt substitution is underlined); and the forward primer used to generate the mutant IRE-ABP proteins was 5'-GACTCACTATAGGGAGACCGGAAGCTTAAT-3'. PCR reactions consisted of wild-type CDM8-IRE-ABP plasmid (0.200 µg) as the template, 200 µM deoxynucleoside triphosphate, 0.21.0 µM primer, 2 mM MgSO4, 10% dimethylsulfoxide, 100 µg/ml BSA, and Pfu DNA Polymerase (12 U) in 1x Pfu DNA polymerase reaction buffer (Stratagene, La Jolla, CA). The reaction cycle included 1.0 min at 94 C, 1.0 min ramp to 52 C, 1.0 min at 52 C, 1.0 min ramp to 72 C, 1.0 min at 72 C, 1.0 min ramp to 94 C, and was repeated 30 times. PCR products were extracted, digested with NcoI and BglII, and then cloned into NcoI- and BglII-cut wild-type CDM8·IRE-ABP from which the corresponding wild-type NcoI/BglII IRE-ABP fragment had been removed.
IRE-ABP cDNA was cloned into NcoI- and HindIII-cut PGEX-GT vector (a kind gift of J. Dixon) to make GST·IRE-ABP (2). The mutant GST·IRE-ABP plasmids were made by digesting the mutant CDM8·IRE-ABP plasmids with NcoI and BglII and subcloning each IRE-ABP fragment with the single nt substitution, into the GST·IRE-ABP plasmid from which the corresponding wild-type NcoI/BglII fragment had been removed. The sequence of the mutant IRE-ABP constructs was confirmed.
The CYP2C12-CAT reporter construct containing CYP2C12 sequences -500 to +23 and the CYP2C11-CAT construct containing CYP2C11 sequences -200 to +26 have been previously described and kindly provided by Dr. C. Legraverend and Dr. A. Ström (13, 14). The min·CAT construct was made by generating a CYP2C11 promoter fragment (nt -65 to +26) by use of PCR with the forward primer 5'-gaattcaagcttCAGAAGCTCATGTTGAATTG and the reverse primer 5'-CATTTTAGCTTCCTTAGCTCCTGAAAATCTCGCCAAGCTC ctcgagatcc (lowercase letters indicate restriction enzyme sites), and the CYP2C11-CAT construct (described above) was used as the template. The site 4·min-CAT construct was made by generating a promoter fragment containing CYP2C12 sequences (-231 to -183) fused to CYP2C11 sequences (-65 to +26) in a PCR reaction using the forward primer 5'-gaattcaagcttGAATGAGTGTATTTATCAATGTTACATGAAATAACTCAATAAACAGAAGCTCATGTTGAATTG (lowercase letters indicate restriction enzyme sites), the same reverse primer used to make the min·CAT construct and the CYP2C11-CAT construct as the template. Both fragments were cut with HindIII/XhoI and used to replace the minimal promoter fragment in the CYP2C11-CAT construct described (14).
The CYP2C12-CAT construct used in Fig. 9 was made by
generating a CYP2C12 fragment (-231 to +23) by PCR with the
forward primer 5'-gaattcaagcttTTAAAGATTTGAATGAGTGT and the reverse
primer, 5'-CATTTTAGCTTCCTTAGCTCCTGAAAATCTCGCCAAGCTCctcgagatcc-3'
(lower-case letters indicate restriction enzyme sites)
using the CYP2C12-CAT construct as the template. The PCR
product was extracted, digested with HindIII and
XhoI, and then cloned into
HindIII/XhoI cut pBLCAT3 that had been digested
with NdeI and HindIII, blunted, and
religated.
The PCR reactions for all reporter constructs contained the following: DNA template (200 ng), 200 µM deoxynucleoside triphosphate, 0.21.0 µM primer, 100 µg/ml BSA and Vent DNA polymerase (1.02.0 U) in 1x reaction buffer from New England BioLabs (Beverly, MA). Reactions were cycled for 1.0 min at 94 C, 1.0 min ramp to 55 C, 1.0 min at 55 C, 1.0 min ramp to 72 C, 1.0 min at 72 C, 1.0 min ramp to 94 C, for 30 cycles. All of the reporter constructs were sequenced by dideoxy-mediated chain termination method with a DNA sequencing kit (United States Biochemical, Cleveland, OH) to verify that promoter sequences were correct. Plasmids used as cotransfection controls were Rous sarcoma virus (RSV)-ß-galactosidase (RSV-ßGAL) and RSV-GH (RSV-GH).
Cell Culture
Hepatocytes were isolated from 8-week-old Sprague-Dawley female
rats (B&K Universal AB, Stockholm, Sweden) that had been maintained
under standardized conditions of light and temperature, with free
access to chow and water (11, 31, 50). Cells were cultured on plastic
at a density of 5 x 106 cells per 100-mm dish in 10
ml DMEM-Hams F12 medium (1:1), supplemented with vitamins (GIBCO BRL,
Gaithersburg, MD), Na2SeO3 (0.1
µM), insulin (0.1 µg/ml), streptomycin (50 U/ml), and
FCS (5%). The serum concentration was reduced to 2% and 0% after
18 h and 42 h, respectively. NIH-3T3 cells were cultured in
DMEM medium supplemented with 10% FBS and 1% glutamine. CHO cells
were cultured in Hams F12 medium supplemented with 10% FBS, 1%
glutamine, and 0.1% gentamicin (G418).
Transfections, Solution Hybridization, and Reporter Gene
Assays
Hepatocytes were transfected by DOTAP (Boehringer Mannheim,
Indianapolis, IN) after 50 h in culture and harvested 15 h
later. C/EBP expression plasmid (3.0 µg) was cotransfected with
increasing amounts of IRE-ABP (1.56.0 µg) or a control plasmid (6.0
µg) to maintain total DNA concentration at 6.0 µg. Solution
hybridization of total nucleic acid was performed from pooled
samples of three transfected dishes, and P450 2C12 and P450 2C11 mRNAs
were analyzed using specific [35S]UTP-labeled
CYP2C11 and CYP2C12 cRNA probes (triplicate
determination) as previously described (11, 31, 50).
NIH-3T3 and CHO cells were plated at a density of approximately 5 x 105 cells per 35-mm dish 24 h before transfection and transfected with a calcium phosphate method (51). Amounts of DNA used for each experiment are indicated in the corresponding figure legends. NIH-3T3 cells were incubated with DNA calcium phosphate precipitates overnight, washed, and incubated in fresh medium. CHO cells were exposed to DNA/calcium phosphate precipitate for 4 h and then shocked with 10% glycerol in 1x PBS for 3 min before the addition of fresh medium. Cells were harvested 48 h after addition of the DNA and CAT assays were performed as described in Ref. 51 .
In NIH cells, RSV-GH was used as a cotransfection control, and GH levels were measured using a RIA kit (Nichols Institute Diagnostic, San Juan, CA). In CHO cells, RSV-ßGAL was used as a cotransfection control, and ß-galactosidase levels were measured by a luminescence assay (Galactolight, Tropix, Bedford, MA).
DNase I Footprinting Analysis
The CYP2C12-CAT construct used in Fig. 2 was digested
with XhoI, labeled with [32P]dCTP, and then
digested with HindIII to release the CYP2C12 DNA
fragment. The CYP2C11-CAT construct used in Fig. 2
was
digested with XhoI, labeled with [32P]dCTP,
and digested with NdeI to release the CYP2C11 DNA
fragment. Purified GST fusion proteins were prepared as described and
cleaved with thrombin to remove GST (2, 51). The proteins were
incubated with 30 x 103 cpm of the labeled
CYP2C12 DNA and CYP2C11 DNA probes for 1 h
in binding buffer: 25 mM HEPES, pH 7.9, 20% glycerol, 78
mM NaCl, 7 mM MgCl2, and 10
mM dithiothreitol (DTT), and 0.02 mg/ml BSA. DNase was
added to the reaction for 1 min, and the reaction was stopped by the
addition of phenol. DNA was purified by phenol/chloroform extraction,
precipitated, and resuspended in dH2O. Equivalent counts
(
2 x 103 cpm) of recovered DNA were loaded on a
6% sequencing gel.
EMSA
Gel shift mobility assays were performed with purified GST
proteins prepared as previously described (2). The proteins were
cleaved with thrombin to remove GST (51). C/EBP and IRE-ABP were
preincubated with antibody for 25 min at room temperature and then
added to the DNA probe (25 x 103 cpm) for 25 min at 4
C in the following buffer: 20 mM HEPES, pH 7. 8, 0.1
mM EDTA, 20% glycerol, 50 mM KCl, 50
mM NaCl, 6.5 mM MgCl2, 5
mM DTT, 0.1 µg deoxyinosinic-deoxycytidylic acid
(dIdC), and 0.1 mg/ml BSA. Reactions were electrophoresed on a
4.5% or 5% nondenaturing polyacrylamide gel in 0.5x TBE (45
mM Tris borate, 1 mM EDTA, pH 8.0) at 170 V for
11.5 h or up to 90 min at 4 C, as indicated in the figure.
Liver nuclear extracts (10 µg) from male rats were prepared as previously described (13). The extracts were preincubated with or without serum (PI or I as indicated in the figures) in a reaction buffer (18 µl) containing 20 mM HEPES, pH 7.8, 0.1 mM EDTA, 20% glycerol, 50 mM KCl, 50 mM NaCl, 5 mM DTT for 25 min at room temperature and then added to a probe mix containing 6.5 mM MgCl2, 0.05 µg dIdC, 5 µg BSA, and DNA probe (2030 x 103 cpm) and further incubated on ice for 25 min. Reactions were subjected to electrophoresis on a 4.5% nondenaturing polyacrylamide gel in TBE at 170 V for 1.52.0 h at 4 C.
Oligonucleotides
The following oligonucleotides were synthesized by Massachusetts
General Hospital Core Facility and used in the EMSAs:
Wild-type CYP2C12 site 4, nts -222 to -196
5'-agcttTATTTATCAATGTTACATGAAATAACTCg
Mutant CYP2C12 site 4, nts -222 to -196
5'-agcttGCCGGCGCCCTGTTACATGCGGCCGCTCg
Wild-type CYP2C11 site A (-129 to -96)
5'-agcttATTTTAACAGGGTCAAGGTCCACAAAGAAGAAATA.
Lowercase letters indicate restriction enzyme sites added for end labeling.
In Vitro Translations and Protein-Protein
Interactions
The pCDNA·C/EBP plasmid used in transfection studies was
also used for in vitro synthesis of proteins in rabbit
reticulocyte lysate using protocols of the supplier (Promega, Madison,
WI). GST pull-down assays were performed using a method described by
Lai et al. (42) with the following modifications.
Glutathione agarose beads (Pharmacia, Piscataway, NJ) were incubated
with GST or GST-fusion proteins. Using one half of the bound beads, the
amount of GST-fusion protein bound to the beads was quantitated by
subjecting proteins released from the beads to SDS/PAGE and staining
the gel with Coomassie blue. The other half of the bound beads (bearing
0.51.0 µg of GST-fusion proteins) were incubated with or without
200 µg/ml ethidium bromide and 35S-labeled in
vitro translated C/EBP
(10 µl) in a solution containing 100
mM NaCl, 1.0 mM EDTA, 20 mM Tris,
and 0.5% NP-40 (NETN) at 4 C for 2 h with constant rotation. The
beads were washed four times with 1 ml of the NETN solution. Bound
proteins were resuspended in SDS sample buffer (51), resolved by
electrophoresis on a 12% SDS polyacrylamide gel, and visualized by
autoradiography.
IRE-ABP Antibody
A chicken polyclonal antibody (1:125,000 by ELISA) was raised
against the full-length GST-IRE-ABP fusion protein. Chicken IgY was
precipitated from egg yolk proteins using polyethylene glycol. The
precipitate was resuspended in PBS and filtered by a 0.2-µm
filter (40 µg of PI and I sera was used in each reaction). A
commercial antibody to C/EBP was obtained (Santa Cruz
Biochemicals, Santa Cruz, CA) and 3.0 µg of antibody were used
in each reaction.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This study was supported by grants from Howard Hughes Medical Institute, the National Institute of Diabetes and Digestive and Kidney Diseases (Grant F31-DK-08729) as well as grants from the Swedish Medical Research Council (Grant 03X-06807), the Karolinska Institute, and the American Scandinavian Foundation.
Received for publication July 23, 1997. Revision received June 9, 1998. Accepted for publication June 12, 1998.
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REFERENCES |
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