(Received for publication, January 26, 1995; and in revised form, May 10, 1995)
From the
The potential of the CREM family of proteins to activate
transcription of the genes encoding the testis-specific isozyme of
angiotensin converting enzyme (ACE) and the gluconeogenic
enzyme, phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (EC 4.1.1.32)
were investigated. Both CREM
and CREM
bind efficiently to the
putative cyclic AMP response element (CRE) present in the ACE
gene (CRET) and to the CRE in the PEPCK gene. In HepG2 cells, the
CRE was required for the strong stimulation by CREM
of the
expression of a chimeric PEPCK (-210 to +73)-chloramphenicol
acetyl transferase (CAT) gene. The CRE could be mutated to the CRET
sequence without losing the stimulatory effects of CREM
. However,
a similar chimeric gene driven by the regulatory region of the
ACE
gene, which contains the CRET site, could only be
stimulated by CREM
when its imperfect TATA element was mutated to
an authentic TATA. Surprisingly, CREM
, an alleged inhibitor of
CRE-mediated transcription, stimulated the expression of both PEPCK-CAT
and ACE
-CAT genes in HepG2 cells, a process which required
the presence of the CRE and the CRET sites, respectively. In contrast,
when the same CRE elements were used to drive the transcription of a
chimeric gene containing the thymidine kinase promoter linked to the
CAT structural gene, CREM
inhibited its expression in HepG2 and
JEG3 cells. The expression of the same chimeric gene, however, was
stimulated by CREM
in F9 embryonal carcinoma cells. These results
demonstrated that the nature of the transcriptional effects of CREM
isoforms on CRE-mediated transcription depends on the specific gene,
the specific cell type and the promoter context of the CRE site.
Angiotensin-converting enzyme (ACE) ()is the key
enzyme of the renin-angiotensin system which regulates blood
pressure(1) . ACE has two isozymic forms. The larger protein,
ACE
, is expressed in vascular endothelial cells, kidney and
intestinal epithelial cells, macrophages and brain
cells(2, 3, 4, 5) . The smaller
isozyme, ACE
, is expressed exclusively in developing sperm
cells(3) . The two isozymes are the products of 5- and
2.5-kilobase mRNAs which are transcribed from the same gene by a
tissue-specific choice of two alternative transcription initiation
sites and two alternative polyadenylation sites in the
gene(6) . Our studies have identified positive and negative
regulatory elements present in the 5`-upstream region of the rabbit
ACE
transcription unit, which are used both in the vascular
endothelial and kidney epithelial cells(7) . This report, on
the other hand, deals with the regulation of ACE
mRNA
transcription.
Inspection of the nucleotide sequence 5` to the
rabbit ACE transcription initiation site revealed several
putative, but no authentic, transcriptional regulatory elements. There
is a TATA-like sequence at -27 and a cyclic AMP response element
(CRE)-like sequence at -52(8) . Further upstream at
-114 and -121, there are sequence motifs similar to one
present in the PGK2 gene which is also expressed in sperm
cells(9, 10) . The ACE
gene contains, in
addition, several palindromic and repeat sequences which may contribute
to its tissue-specific expression(8) . Experimental analysis of
the functional roles of these putative sites has, however, been
difficult mainly because of the unavailability of in vitro culture systems of the relevant cell types which can be used for
transfection of reporter genes and measurements of their resultant
expression. To circumvent these problems, transgenic assays have been
used. Mice carrying reporter transgenes driven by putative regulatory
regions of the ACE
gene have been tested for reporter gene
expression in the testis. Such an analysis with the rabbit gene has
indicated that a region containing 325 bp 5` to the ACE
transcription start site is sufficient for driving sperm-specific
expression of the reporter gene. (
)Similar analysis with the
mouse gene has narrowed down the regulatory region to 91 bp upstream of
the transcription start site(11) . There are two putative
regulatory sites in this region, a CRE-like sequence, CRET, and a
TATA-like sequence. We have examined the transcriptional roles, if any,
of these elements using a heterologous cell culture system.
Our
transfection analyses was performed using the HepG2 cell, a hepatoma
cell line which has been widely used for studying transcriptional
regulation of cAMP-responsive genes. One such gene encodes the
gluconeogenic enzyme, PEPCK. The transcriptional regulation of the
PEPCK gene has been extensively studied by us (12, 13, 14) and
others(15, 16) . We have shown that a chimeric
PEPCK-CAT gene containing a region from -210 to +73 of the
PEPCK promoter is responsive to cAMP in hepatoma cells(14) . A
CRE present in this region mediates the major portion of the cAMP
response(13, 17) . Several trans-acting factors can
bind to this element and activate
transcription(14, 18, 19) . In the current
study, we have analyzed the potential role of CREM and CREM
in the regulation of PEPCK gene transcription in HepG2 cells and have
compared the results of this analysis with the induction of
transcription of the gene for ACE
.
Since the CRE-like
site may be involved in sperm-specific regulation of ACE expression, we were also interested in determining the
trans-acting factors which are preferentially expressed in sperm cells
and bind to this regulatory element. One such family of proteins, the
CREM proteins, has been recently described(20) . This family of
proteins originates from the same gene by cell-specific alternative
splicing. They all contain DNA-binding domains, which recognize the
CRE, and a phosphorylation box which is the target of phosphorylation
by cAMP-activated PKA. Some isoforms such as CREM
contain, in
addition, glutamine-rich domains which are absent from other isoforms
such as CREM
. CREM
is a strong activator of transcription,
which is cAMP dependent and mediated by the CRE. On the other hand,
other forms lacking the glutamine-rich domain, such as CREM
, have
been shown to repress CRE-mediated transcription(21) . Thus,
the relative levels of CREM
and CREM
can control the extent
of cAMP-mediated gene transcription in cell types which express these
proteins. Both activator and repressor isoforms of CREM are found at a
low level in immature sperm cells. But during later stages of sperm
differentiation, there is a pronounced isoform switch so that a large
level of CREM
, the activator form, is
produced(22, 23) . Since ACE
is expressed
in maturing sperm, CREM
could be a strong candidate for promoting
its transcription. This hypothesis was tested in the current study.
Our studies demonstrated that CREM stimulated transcription via
both the CRE of the PEPCK gene and the CRET of the ACE
gene. Expression of the latter gene in HepG2 cells required, in
addition, mutation of the TATA-like sequence to an authentic TATA.
Unexpectedly, CREM
also stimulated transcription of both genes.
Further experiments demonstrated that the ability of CREM
to
stimulate or repress gene transcription depends on the promoter context
of the CRE sequence and the cell type.
Site-directed mutagenesis of the PEPCK-CAT and ACET-CAT vectors was performed using the Muta-Gene® phagemid in vitro mutagenesis system from Bio-Rad, strictly following the manufacturer's instructions. All mutations of the PEPCK gene promoter were performed in the single stranded replicative form of the PTZ18R derived PEPCK-CAT expression vectors, as described previously(12, 24) . -210CRE2 mCREmPEPCK-CAT and -210CRE2 mACETPEPCK-CAT constructs were thus obtained by using mutagenizing oligonucleotides, CRE1 mTYGS and CRETOACET, on the -210CRE2 mPEPCK-CAT background, respectively. Mutagenesis of -85ACET gene promoter was performed by subcloning this gene segment in the pBlueScript(KS) vector. TATAm oligonucleotide was used to generate the -85ACETTATAm vector. All subcloning reactions were performed using directional cloning strategies and were confirmed by multiple restriction digestion analysis. All site-directed mutations were confirmed by sequencing using the dideoxy sequencing reaction.
The chimeric TK-CAT gene contains a -109 to +51 segment of the herpes simplex virus thymidine kinase gene (where transcription state site is designated as +1), ligated upstream of the CAT gene. To generate the CRETK-CAT and CRETTK-CAT plasmids, blunted double-stranded 18-bp oligonucleotides containing the CRE and CRET sequences were cloned 5` to the TK promoter in the blunted BamHI site of the TK-CAT vector. Plasmids with single copies of CRE and CRET sequences inserted at the correct position and orientation were selected by sequencing several clones.
Double-stranded CRE, CRE2, CREmut, and CRET probes were prepared by annealing synthetic complimentary oligodeoxynucleotides followed by enzymatic filling-in of the shorter strands: ACETCRESEN, GGCCCCTTACGTCAGAGG; ACETCREAS, CCTCTGACGTAAGGGGCC; CRE2S, GGCCGGCCCCTTAGGTCAGAGGCGAGC; CRE2AS, GCCTCTGACCTAAGGGGCC; CREmTYGS, GGCCGGCCCCCTGCGGACAGAGGCGAGC; CREmTYGAS, GCCTCTGTCCGCAGGGGCC; CRE1TOACET, GGCCGGCCCCCTGAGGTCAGAGGCGAGC; ACEtAS, GCCTCTGACCTCAGGGGCC.
Figure 1:
Gel
mobility shift assay to determine the different affinities of CREM
for the different CRE sequences. A radiolabeled 18-bp oligonucleotide
(
40,000 cpm, 50 pg) corresponding to the CRE sequence from the
PEPCK gene was incubated in the absence or presence of various
reticulocyte lysate protein extracts (2 µl) and various molar
excesses of cold competitor probes. Lane 1, the probe alone; lane 2, the probe and reticulocyte lysate; lane 3,
the probe and in vitro translated CREM
; lanes 4 and 9, the probe and in vitro translated
CREM
; lanes 5-8, the probe, CREM
and 20-,
100-, 400, and 2,000-fold molar excess of the unlabeled CRE
oligonucleotide; lanes 10-12, the probe, CREM
and
400-, 3,000-, and 30,000-fold molar excess of unlabeled CREmut; lanes 13-15, the probe, CREM
and 400-, 2,000-, and
20,000-fold molar excess of unlabeled CRET; lanes 16-18,
the probe, CREM
and 400-, 2,000-, and 20,000-fold molar excess of
unlabeled CRE2. Arrows A, B, C, and D indicate the specific DNA-protein
complexes.
Figure 2:
Transcription stimulation by CREM via
the CRE sites of PEPCK gene. A, schematic diagram of the
PEPCK-CAT promoter from -210 to +73 positions, where +1
is the transcription start site. Important protein binding domains
within this region of the PEPCK gene are outlined above the promoter. B, HepG2 cells were transfected with plasmids containing the
PEPCK-CAT with serial 5` deletions in the PEPCK promoter starting at
positions indicated below the bars. 5 µg of PEPCK-CAT
plasmid were transfected alone (open bar); cotransfected with
5 µg of CREM
expression vector (striped bar);
cotransfected with 5 µg of an expression plasmid for the C subunit
of PKA (hatched bar); cotransfected with SR
-PKA and
CREM
expression plasmids (solid bar). Each bar represents
the mean value from a duplicate experiment, and the dot over the bar
shows the higher value. Each experiment was performed at least three
times using at least two different plasmid DNA preparations and batches
of cells. C, transfections were carried out in HepG2 cells
using 5 µg of -210PEPCK-CAT plasmids containing specific
mutations in the protein binding domains of the promoter. The sites
which were block mutated are indicated on the horizontal axis. Open
bar, cotransfected with 5 µg of SR
-PKA; solid
bar, 5 µg of SR
-PKA and 5 µg of CREM
. Data
analysis and presentation are similar to those described for B.
Since the region of the PEPCK promoter between
-210 and +73 contains several well characterized cis-acting
binding sites (Fig.2A), a series of 5`-deletion
mutants were tested to identify the site responsible for the mediation
of CREM stimulation. The PEPCK promoter deleted to -174
(-174 to +73) lacks the HNF-1 binding site and was
stimulated by the C subunit of PKA and CREM
to levels very similar
to those seen with the PEPCK promoter deleted to -210 (Fig.2B). The region of the PEPCK promoter between
-174 and +73 contains two putative CRE sites (CRE and CRE2)
and an NF-1 binding site. Further deletion to -109 upstream
position of PEPCK promoter decreased its basal activity to about 15% of
that noted with the promoter deleted to -210 but a pronounced
stimulation was still observed with the cotransfection of CREM
.
The PEPCK promoter deleted to -109 contains only the CRE which
has previously been shown to bind CREB, Fos/Jun heterodimers and
members of C/EBP family of transcription factors. These results suggest
that the CRE is sufficient for mediating the effect of CREM
on
transcription from the PEPCK promoter.
To further elucidate the role
of the various cis-elements, mutants of these sites in the PEPCK
promoter bretween -210 and +73 were tested. When the CRE2
site was mutated, there was a negligible effect on transcription from
the PEPCK promoter in HepG2 cells (Fig.2C). Similarly,
mutation of the NF-1 site did not alter either the basal level of
transcriuption from the PEPCK promoter or the CREM-induced level
of expression. Mutation of the CRE, on the other hand, decreased both
levels drastically. Since transcription was stimulated to a slight
extent with the CRE mutated, it is possible that at least some effects
of CREM
on PEPCK gene expression is mediated through the CRE2
site. This observation is consistent with the observed weaker but
detectable binding of CRE2 to CREM
in gel shift assays (Table1). When both CRE and CRE2 sites were mutated, the
response to CREM
was completely abrogated.
Figure 3:
CREM can stimulate transcription
through CRET. HepG2 cells were transfected with 5 µg of
-210PEPCK-CAT vectors containing specific mutations.
-210CRE2 mPEPCK-CAT vector contains a disruptive mutation in the
CRE2 binding site. In the -210CRETCRE2 mPEPCK-CAT vector, in
addition to the CRE2 mutation, the CRE site was mutated to a CRET
binding sequence. The -210CREmutCRE2 mPEPCK-CAT vector contains
mutations of both the CRE and CRE2 sites. The CAT reporter plasmids
were cotransfected along with 5 µg of SR
-PKA expression vector
alone (open bars); or with 5 µg each of PKA and CREM
expression vectors (solid bars). Data analysis and
presentation are similar to those described for Fig.2B.
Once we established that CRET can mediate
transcriptional stimulation by CREM in the PEPCK gene context, we
tested if the same is true in context of the ACE
gene. For
this purpose, we used a reporter CAT gene driven by 85 bp of the
transcriptional regulatory region of the ACE
transcription
unit (Fig.4A). This region contains the CRET site at
-52 position and a putative TATA element at -26 position.
The corresponding region from the mouse ACE
transcription
unit, which has strong sequence homology to the rabbit gene used in our
study, has been shown to drive the expression of a reporter gene in the
sperm cells of adult transgenic mice(11) . We wanted to test if
CREM
, which is known to be expressed in the sperm cells, could be
the responsible transcription factor. For this purpose, the
ACE
-CAT gene and the CREM
expression vector were
cotransfected in HepG2 cells which do not express the gene for this
transcription factor. CREM
failed to stimulate the expression of
this chimeric gene, not only in HepG2 cells (Fig.4B)
but also in HeLa and NIH3T3 cells (data not shown). The ACE
promoter lacks an authentic TATA sequence but instead contains a
TATA-like sequence at -27 position; we determined whether the
lack of responsiveness to CREM
could be due to the defective TATA
element in the ACE
-CAT gene. To test this hypothesis, its
TCTTATT sequence was mutated to TATAATT. The authentic TATA mutant of
the ACE
-CAT gene responded to CREM
stimulation very
efficiently (Fig.4B).
Figure 4:
CREM can stimulate the transcription
of ACE
gene in somatic cells only after mutation of its
TATA-like element to an authentic TATA sequence. A, sequence
of the upstream region of the ACE
transcription unit from
position -85 to +30 (transcription start site is designated
+1) which was used to construct the ACE
-CAT gene. The
potential protein-binding sites are underlined. B,
transfections were carried out in HepG2 cells using 5 µg of an
expression plasmid containing the ACE
-CAT gene expression
plasmids. ACE
TATAm-CAT vector contains a mutation whereby
the native TATA-like element of the ACE
gene has been
mutated to an authentic TATA sequence. Cotransfections were performed
with either 5 µg of expression vector containing the SR
-PKA
gene (open bars) or with 5 µg each of the expressions
vectors for the catalytic subunit of PKA and CREM
(solid
bars). C, same experiment as in B, performed in
the ACEp-producing OPK cells. An additional control was 5 µg of CAT
reporter plasmid transfected alone (hatched bars). Open and solid bars represent the same conditions as described
in B. Data analysis and presentation are similar to those
described for Fig.2B.
These results suggest that in
somatic cells, such as HepG2, the ACE transcription unit
cannot be expressed even if a positive transcription factor such as
CREM
, which binds to the CRE, is made available. It is possibly
because the transcriptional machinery in these cells do not recognize
the TATA-like sequence present in this gene. Thus, the tissue-specific
expression of the ACE
mRNA is influenced by both the CRET
element and the TATA-like element which apparently is used in the
transcription machinery in sperm cells. In the experiment shown in Fig.4C, we examined the situation in cell types in
which the ACE gene is transcriptionally active but only the ACE
transcript is synthesized. OPK cells, a kidney cell line,
expresses ACE
but not ACE
(7) . In those
cells, ACE
-CAT was again not stimulated by CREM
unless
the TATA-like element was mutated to an authentic TATA (Fig.4C). These results indicate that the
tissue-specific expression of ACE
is, at least partly,
dictated by the defective TATA element not only in cells which do not
express the ACE gene at all but also in cells which contain the
ACE
mRNA, but not the ACE
mRNA.
Figure 5:
Transcriptional activation of ACE gene by CREM
. The effect of cotransfection of CREM
expression vector on the transcription of a chimeric
ACE
TATAm-CAT gene was tested in HepG2 cells. Five µg of
ACE
TATAm-CAT reporter plasmid was cotransfected with 5
µg of SR
-PKA and with 5 µg of either pUC18 plasmid (open bar), CREM
expression plasmid (hatched
bar), or CREM
expression vector (solid bar). Data
analysis and presentation are similar to those described for Fig.2B.
Since CREM can bind to the PEPCK CRE
sequence better than the CRET sequence of ACE
gene (Table1), we tested if CREM
could stimulate transcription
of the PEPCK-CAT gene. Indeed, there was strong stimulation of CAT
expression in response to CREM
(Fig.6). Although somewhat
weaker, the overall pattern of stimulation was similar to that observed
with CREM
. The stimulation was enhanced by PKA and mediated mostly
by the CRE site of the PEPCK promoter. These results clearly
demonstrated that CREM
can act as a transcriptional activator in
the appropriate cellular context.
Figure 6:
Transcriptional activation by CREM. A, HepG2 cells were transfected with the PEPCK-CAT plasmids
with serial 5` deletions starting at positions indicated below the
bars. 5 µg of PEPCK-CAT plasmid was transfected alone (open
bar); cotransfected with 5 µg of CREM
expression vector (striped bar); cotransfected with 5 µg of SR
-PKA (hatched bar); cotransfected with SR
-PKA and CREM
expression plasmids (solid bar). B, transfections
were carried out in HepG2 cells using 5 µg of -210PEPCK-CAT
plasmids containing specific mutations in the protein binding domains
of the promoter. The site which was mutated is indicated on the
horizontal axis. Open bar, cotransfected with 5 µg of
SR
-PKA; solid bar, 5 µg of SR
-PKA and 5 µg
of CREM
. Data analysis and presentation are similar to those
described for Fig.2B.
Figure 7:
Promoter context-dependent effects of
CREM. The effect of CREM
on transcription of a chimeric
TK-CAT gene was tested in JEG-3 and HepG2 cell lines. The thymidine
kinase promoter (-151 to +60) was cloned upstream of the CAT
reporter gene to obtain TK-CAT plasmid. CRETK-CAT and CRETTK-CAT
vectors contain the CRE sequence from the PEPCK gene and the CRET
sequence from the ACE
gene subcloned 5` to the TK promoter,
respectively. A, JEG-3 cells were transfected with the TK-CAT,
CRETK-CAT, and CRETTK-CAT plasmids, as indicated below the bars. Open bar, 5 µg of CAT plasmid was transfected alone; striped bar, cotransfected with 5 µg of CREM
expression vector; hatched bar, cotransfected with 5 µg of
SR
-PKA; solid bar, cotransfected with 5 µg each of
SR
-PKA and CREM
expression plasmids. B, similar
experiment using CRETK-CAT and CRETTK-CAT plasmids was performed in
HepG2 cells. Symbols for the bars are the same as in A. Data
analysis and presentation are similar to those described for Fig.2B.
Possible contributions by the cellular contexts
to the observed stimulatory and repressing activities of CREM were
further explored by testing the effects of CREM
and CREM
on
the expression of the PEPCK-CAT gene and the CRETK-CAT gene in F9
embryonal carcinoma cells. F9 cells, in contrast to the other cell
lines used in this study, do not contain a detectable level of CREB
with which CREM
is known to be able to form
heterodimers(35) . Neither CREM
nor CREM
could
stimulate the expression of the PEPCK-CAT gene in F9 cells, even in the
presence of the catalytic subunit of PKA (Fig.8). This was
possibly due to the known tissue specificity of the expression of this
gene which is controlled by sites other than the CRE. The CRETK-CAT
gene was, however, expressed at a low, but detectable, level in the
presence of PKA. The level of expression was enhanced by about 10-fold
in the presence of CREM
and by about 3-fold in the presence of
CREM
. Thus, expression of the same reporter gene, CRETK-CAT, could
either be stimulated or be repressed by CREM
in different cell
lines suggesting that its interaction with other transcription factors
may be a crucial determining factor.
Figure 8:
Effects of CREM in F9 cells.
Expression of PEPCK-CAT and CRETK-CAT genes was tested in F9 cells. 10
µg of the reporter gene were cotransfected with 5 µg of
SR
-PKA gene (open bar), with 5 µg of SR
-PKA and
5 µg of CREM-
(solid bar), or with 5 µg of
SR
-PKA and 5 µg of CREM
(hatched bar). Data
analysis and presentation are similar to those described for Fig.2B.
The present study provides useful information regarding the
possible basis of tissue-specific expression of ACE in the
testis. Such a tissue-specific expression has two layers of complexity.
In the majority of tissues neither isoform of ACE is expressed at all.
It is quite possible that the ACE gene is buried in transcriptionally
inactive parts of the chromosome in these tissues. However, there are
other tissues such as kidney, vascular endothelial cells, and
macrophages in which one isozyme, ACE
, is exclusively
expressed, whereas in sperm cells only the other isozyme,
ACE
, is expressed. The ACE gene cannot be in a
transcriptionally inactive environment in these tissues. Our studies
indicate that two cis-elements of the ACE
transcription
unit mediate its tissue-specific expression. The CRET site was shown to
be capable of serving as a cAMP-inducible enhancer. The trans-acting
factors responsible for mediating this response could be members of the
CREM family of transcription factors. Our in vitro transfection expression clearly demonstrated that CRET can bind
and respond to CREM
, a potent trans-activator of transcription.
Previous studies have established that CREM proteins are expressed in
testis. Moreover, spermatogenesis is accompanied by a isoform switching
of CREM. CREM
is abundant in maturing sperm cells, the site of
ACE
synthesis(22, 23) .
Our studies
also indicate another possible tissue-specific cis-element, the
TATA-like sequence present in the ACE gene. In the two
somatic cell lines used in our study, one, a nonexpressor of ACE, and
the other, an expressor of ACE
, ACE
upstream
region could only drive transcription after the TATA-like sequence was
converted to an authentic TATA element. Thus, in these cells, both the
availability of CREM and the presence of authentic TATA were necessary
for the expression of ACE
-reporter genes. This may explain
the lack of expression of ACE
in several cell types, other
than sperm, in which CREM
is highly expressed. Presumably there is
a specific transcription factor in ACE
-expressing sperm
cells which recognizes the TATA-like element present in ACE
gene. The existence of such a cell type and sequence-specific
TATA-binding factor remains to be documented. Alternatively, it is
possible that some other as yet unrecognized cis-element impacts the
sperm-specificity. In this scenario, the TATA-like sequence is
irrelevant and noncontributing and the ACE
gene is driven
by a TATA-less promoter like many other known genes(29) .
Testing the ability of appropriately constructed reporter genes to be
expressed in sperm cells will be necessary for providing definitive
answers. Such experiments using transgenic mice are currently in
progress.
Our experiments defined the spectrum of interaction
between CREM proteins and different CRE sequence. The CREmut sequence
which varies from the canonical CRE sequence present in the
somatostatin gene, TGACGTCA, in positions 3, 4, and 6, failed to bind
to CREM and CREM
and did not promote transcription in
transfection analysis (Table1). The CRE from the PEPCK gene,
which varies only in position 2 from the somatostatin CRE, was highly
efficient in both binding CREMs and mediating CREM-driven
transcription. CRET, which also varies in only one position, 4, was
less efficient than CRE1 by both measures. The cryptic CRE2 from the
PEPCK gene, which combines the differences of CRE1 and CRET and varies
from the somatostatin CRE at both positions 2 and 4, was even less
efficient and it could barely mediate CREM-driven transcription. As
expected from their structures, the binding properties of CREM
and
CREM
were parallel although the absolute affinities were
different.
The most unexpected observation reported here is the
stimulation of CRE-mediated transcription by both CREM and
CREM
. Furthermore, the observed difference in the stimulatory and
the inhibitory phenotypes of CREM
, in different cell types or when
the same CRE sequence is present in different promoter contexts,
indicates a novel mode of regulation of transcription. Similar
dichotomy has been previously noted for the chicken ovalbumin upstream
promoter-transcription factor which represses the promoter of the
ornithine transcarbamylase gene but activates the promoters of several
other genes(30) . Consequently, it should be appreciated that
conclusions drawn from studying the transcriptional regulation of one
gene may not be valid for another gene even if the same cis-element and
the same trans-acting factor are involved in regulating the
transcription of both genes.
CREM has previously been
identified as a repressor of CRE-mediated transcription. The
experiments leading to this characterization were generally carried out
using artificial promoters containing the CRE of the somatostatin gene
and the promoter of the thymidine kinase gene, which is similar to the
chimeric promoters we used in the experiment shown in Fig.7.
The observed repression of transcription by CREM
was rationalized
by its structural difference as compared to CREM
; CREM
does
not contain the glutamine-rich domains present in CREM
. Such
domains can serve as transcriptional activators and they often exist in
transcription factor motifs distinct from the DNA-binding and other
regulatory domains. Although CREM
lacks the glutamine-rich
domains, its DNA-binding domains are identical to those of CREM
.
Moreover, both isoforms share the same phosphorylation box or
kinase-inducible domain. Within this domain is serine 117 which is the
target of phosphorylation by not only by the C subunit of PKA but also
by PKC, calmodulin kinase, p34
and p70
(31) . How phosphorylation of this residue promotes the
transcriptional activation potential of CREM proteins is not known.
However, more is known about the activation mechanism of another
CRE-binding transcriptional activator, CREB, upon its PKA-mediated
phosphorylation. Such a phosphorylation does not change the affinity of
the protein for its cognate DNA element(16, 32) , but
changes its affinity for the CREB-binding protein, CBP, which serves as
a co-activator of transcription(33) . The kinase-inducible
domain (KID) of CREB is also present in CREM
and CREM
. Thus,
it is not unlikely that PKA-activated CREM
and CREM
also bind
CBP and promote transcription as a result of this interaction. Using
engineered genes, it has been shown that KID can act as a conditional
activator working either through a physically attached activation
domain or through a promoter-bound protein in trans(34) . Here, we have uncovered a natural example
of these two modes of transactivation using the regulatory regions of
the PEPCK and ACE
genes. PKA-dependent activation by
CREM
probably depends on the phosphorylation of KID, resultant
binding of CBP or another protein having equivalent properties, and its
interaction with the glutamine-rich activation domains present in the
CREM
itself. In the case of CREM
, the first two steps might
be the same but the bound CBP, instead of interacting with
glutamine-rich domains which are absent for CREM
, interacts with
neighboring factors bound to other cis-elements present in the gene.
This model would postulate that the last crucial protein-protein
interaction between CREM
-bound CBP and other proteins bound to the
transcription unit is different when the PEPCK CRE sequence is present
in its natural context versus when it is present in the
context of the TK promoter. The exact nature of this difference remains
to be identified.
The observed differences in the effects of
CREM could also, in principle, be due to a difference in the
extent of its hetrodimerization with other CRE-binding factors such as
CREB(35) . The observed stimulation of expression of the
CRETK-CAT gene by CREM
in F9 cells rules out an involvement of
CREB in this process, since these cells are devoid of this
transcription factor. It appears therefore that CREM
, like
CREM
, can by itself promote transcription through a CRE site
present in the right genetic context in the right type of cell.