Institute for Hormone and Fertility Research University of Hamburg 22529 Hamburg, Germany
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ABSTRACT |
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INTRODUCTION |
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The PRL gene in the human endometrium is transcribed from an
alternative promoter located 6 kb upstream of the pituitary PRL
promoter (15). This decidual-type PRL (dPRL) promoter is not responsive
to progesterone but is cAMP-inducible (16). We have shown previously
that ES cells, decidualized by long-term treatment with RLX and
progestin, do not desensitize to the persistent cAMP stimulus. The
protein for the regulatory subunit of PKA, RI, is down-regulated
whereas the level of catalytic subunit remains unaltered, resulting in
an increase in free catalytic subunit (17). Here we investigated the
involvement of downstream mediators of the cAMP signal, i.e.
the transcriptional modulators CREM (cAMP response element modulator)
and ICER (inducible cAMP early repressor). CREM was identified as a
modulator of cAMP response element binding protein (CREB) action (18).
CREB binds to cAMP response element (CRE) sequences and activates gene
transcription upon phosphorylation (19). CREM can form heterodimers
with CREB or compete for the binding site as a homodimer (20, 21, 22). The
CREM gene shares regions of high homology with the
CREB gene and probably arouse by gene duplication (19, 23).
CREM expression is regulated at multiple levels to give rise to both
activators and inhibitors of PKA-stimulated transcription. First,
numerous isoforms are generated by alternative splicing of exons
encoding functional modules (20). These modules include two alternative
DNA binding and dimerization domains (DBD I and II), a phosphorylation
domain (P box), and two glutamine-rich regions conveying activator
function (Q1, Q2). CREM isoforms lacking Q domains are transcriptional
repressors, e.g. CREM-
and CREM-
, which differs from
the former by the absence of the small
-exon (18). The various
CREM-
forms, including either one or both Q domains, are
transcriptional activators and are significantly up-regulated during
spermatogenesis (22, 24). At a second level, alternative
translation initiation on a CREM-
transcript can give rise to the
shorter inhibitory form S-CREM, as has been demonstrated in the mouse
brain (25). Finally, complexity is added to CREM expression by the use
of an alternative cAMP-inducible intronic promoter (P2) upstream of the
DBDs to generate the inducible early repressor ICER (26) (Fig. 1
). P2 carries a cluster of four CREs that mediate cAMP
induction of ICER expression. ICER protein consists of no other
functional domain than a DBD and acts as a repressor on cAMP-responsive
promoters including its own promoter, thereby establishing a negative
autoregulatory loop (27). This mechanism has been implicated in the
circadian rhythmicity of cAMP responsiveness in the neuroendocrine
system (26).
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RESULTS |
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The function of CREM isoforms is dictated by their modular
composition as a consequence of alternative splicing. We wanted to
determine which isoforms were expressed in human ES cells.
Amplification of the 5'-portion of CREM, upstream of the two
alternative DBDs, with primer pair C-H (see Fig. 1) yielded multiple
products, indicating that alternative splicing involving modules D to G
takes place (Fig. 2D
). Amplification of the entire sequences using
primer pair C-J indicated that both DBDs are used, because isoforms
expressing DBD II are 399 bp shorter (lacking module I) than those
expressing DBD I. The latter forms include module I, leading to
translation termination at the stop codon immediately after DBD I.
Hybridizations with module-specific probes revealed no change in the
level of expression of any of the CREM isoforms between
undifferentiated and differentiated cells (Fig. 2D
). Probe H visualized
all isoforms. Probe G detected a subpopulation that includes exon
and therefore is 36 bp larger than the corresponding forms lacking this
exon. Hybridization with the module-specific probes E and F revealed
the presence of the P box or the Q2 domain in some, but not all,
isoforms. The Q1 domain was not present in any of the transcripts
inasmuch as probe D yielded no signal (not shown).
Cloning of CREM- and ICER-Isoforms from Human ES Cells
The size of some CREM isoforms was not compatible with the sum of
any of the combinations of the known modules. We therefore performed
PCR with the primers B and J anchored in the 5'- and 3'-untranslated
region (UTR) of CREM, respectively, to obtain full-length coding
regions (Fig. 3). The resultant pattern was similar to
that shown for primer pair C-J in Fig. 2D
. Products between 0.8 and 1.3
kb in size were isolated and cloned. Six different populations of
clones were obtained based on size and restriction mapping, all
including DBD I but none containing exon
(Fig. 3
). Clone CREM-22
represented the known inhibitor CREM-
whereas all other forms have
not yet been described. CREM-10 is a potential activator as it includes
P box and Q2 domain but, in contrast to the established CREM-
forms,
it contains the first of the two DBDs and was therefore termed
CREM-
2
. CREM-17 lacks Q domains and P box
(CREM-
1,P, 2). In general, the absence or presence of the Q domains
or of exon
does not affect the open reading frame (ORF), since
their nucleotide numbers represent multiples of 3. However, the
presence of the P box (module E; 241 nucleotides) is required because
it contributes one additional nucleotide to complement the incomplete
codon -CT at the 5'-end of module H. The alternative splicing, leading
to omission of the P box (module E) in CREM-17, was therefore expected
to interrupt the ORF. Sequencing of the splice junction revealed an
additional irregularity due to a missing nucleotide at the 3'-end of
module C (Fig. 4
). However, the ORF remains disrupted
because an in-frame stop codon is encountered soon after entry into
module H. Likewise, in the isoform CREM-20, which is lacking Q1 and P
box (CREM-
1,P), a stop codon appears immediately after entry into Q2
(module F) (Fig. 3
).
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None of the constructs affected basal activity of the reporter constructs in the absence of PKA expression vector (not shown), suggesting a function linked to PKA-induced nuclear events. To further exclude that the CREM expression vectors, particularly those carrying in-frame stop codons upstream of the DBD (pRc/CREM-17, -20, -23, -24), inhibited production of reporter protein nonspecifically, we tested these constructs in a different, PKA-independent system. SKUT-1B cells were transfected with the Pit-1 responsive reporter gene construct mPit-0.3/luc, which carries 0.3 kb 5'-flanking DNA of the mouse Pit-1 gene linked to the luciferase reporter gene (30). Cotransfection of a Pit-1 expression vector, pCMV-hPit1 (30), caused a 4-fold induction of reporter gene activity, which was not altered by introduction of any of the CREM or ICER expression vectors (data not shown).
Novel CREM Protein Isoforms Are Generated by Internal Translation
Initiation
The ambiguity in the function of overexpressed CREM-10 indicated
that both negative and positive modulators were translated from the
transcript, which could be explained by translation initiation both at
the first (or closely adjacent second) AUG in module C (see Fig. 8C, methionine residues Met 1 and Met 2) and at a
downstream AUG. The same alternative start codon that gives rise to
S-CREM from the murine CREM-
mRNA is conserved in the human CREM
sequence in module E (adjacent to the P box within a Kozak context, ACC
AUG G) (31) (Fig. 8C
, Met 3). The fact that CREM-17, -20,
-23, and -24 were efficient inhibitors of PKA-stimulated gene
transcription suggested that functional protein was translated from
these transcripts despite the short 5'-ORF. Several in-frame AUG codons
are present in these transcripts downstream of the stop codons
indicated in Fig. 3
. The AUGs shown in Fig. 8C
in module F (Met 4) and
at the 5'- and 3'-ends of module H (Met 5, Met 7) are in a rather poor
initiation context, whereas the Met 6 in module H (GUG AUG
G) complies with the Kozak rules.
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CREM, S-CREM, and SS-CREM Isoforms Homo- and Heterodimerize and
Bind to a CRE
The FLAG-tagged proteins obtained by in vitro
transcription/translation as described above were used for
electrophoretic mobility shift assays (EMSA) on a consensus CRE probe
(Fig. 9). Two retarded complexes, which were competed by
excess unlabeled CRE but were not affected by the addition of FLAG
antiserum, were seen with translation product from the empty vector
(pRc/CMV) and represent CREBs endogenous to the
transcription/translation cocktail. All preparations resulting from
CREM/FLAG cDNAs yielded additional complexes that were quantitatively
supershifted by FLAG antiserum. CREM-10 yielded
CREM-
2
and S-CREM-
2
homodimers and
an intermediate heterodimer. CREM-17 generated SS-CREM-
homodimer.
CREM-20 gave rise to a prominent SS-CREM-
2
homodimer
and a second smaller complex probably resulting from heterodimerization
with a limited amount of SS-CREM-
. CREM-22 generated CREM-
and
S-CREM-
homodimers and an intermediate heterodimer.
SS-CREM-
2
homodimer was obtained with CREM-23; this
complex caused a higher retardation than that of CREM-20-based
SS-CREM-
2
homodimer, again due to the C-terminally
shifted stop codon in CREM-23/FLAG. Finally, with CREM-24, only
S-CREM-
homodimer was formed.
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To examine whether short CREM isoforms also heterodimerized with
CREB, we produced FLAG-tagged S-CREM- protein by in vitro
transcription/translation of CREM-24/FLAG. Upon EMSA, this protein
forms a single complex with labeled CRE, as already shown in Fig. 9
. We
then added the DBD of CREB (human CREB bZIP, amino acids 254327,
10 kDa), which results in a faster migrating complex. Coincubation
of S-CREM-
and CREB led to the formation of an intermediate complex
that represents a CREM/CREB heterodimer because this complex and the
S-CREM-
homodimeric complex were supershifted by FLAG antibody,
whereas CREB homodimer remained unaffected (Fig. 10
).
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To assess the subcellular localization of the CREM protein isoforms as
summarized in Fig. 8, we applied immunofluorescent staining to SKUT-1B
cells transfected with the CREM/FLAG constructs. FLAG antibody detected
FLAG-tagged proteins produced from all constructs and localized these
proteins almost exclusively to the nuclei of the transfected cells
(Fig. 12
). Equivalent results were obtained using
either polyclonal FLAG antiserum or M2 monoclonal FLAG antibody, which
detects C-terminally inserted FLAG epitope.
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DISCUSSION |
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All alternatively spliced forms of CREM described so far retain the
coding sequence in frame (19). In contrast, numerous exons have been
identified in the CREB gene (reviewed in Refs. 19 and 20):
,
,
(29), W (36), Y (37), and Z (38) which, when inserted
into the CREB transcript, introduce in-frame stop codons upstream of
the DBD. The function of such transcripts remained elusive, as
carboxy-terminally truncated proteins would be lacking the DBD and the
nuclear translocation signal that resides within the basic region of
the CREB DBD. Only recently the polycistronic nature of CREB
transcripts containing exon W was demonstrated in that internal
translation initiation in exons W and H could lead to the formation of
short transcriptional repressors, I-CREB(l) and I-CREB(s),
expressing the DBD, in testes (38, 39). Expression of these repressors
is potentially involved in cAMP-dependent regulatory pathways during
spermatogenesis.
Here we report for the first time a similar multilevel complexity in
the expression of transcriptional repressors from the CREM
gene, involving both alternative splicing and alternative translation
initiation. Upon cloning of CREM transcripts from human ES cells, we
identified a novel exon residing about 4 kb downstream of the first
protein-coding exon and 5' to the sequence encoding the transactivation
domain Q1. CREM exon
resembles CREB exon
both by its genomic
location (exon
is separated from the first protein coding exon of
the CREB gene by a 5.7-kb intron) and by the presence of
in-frame stop codons (29); exons
of the CREB and of the
CREM gene, however, share no sequence homology. Exon
was
found in several transcript isoforms; in the cDNA clones CREM-23 and
CREM-24 isolated from human ES cells it was either spliced to the Q2
domain (CREM-
1,P) or to the first exon coding for the P box
(CREM-
1,2). In addition to these CREM-
isoforms, we isolated
previously undescribed splice variants lacking the P box in which the
first protein-coding exon was either spliced to the Q2 domain (clone
CREM-20, termed CREM-
1,P) or directly to the exon encoding the
5'-portion of DBD I (clone CREM-17, termed CREM-
1,P, 2). All these
novel isoforms appeared nonfunctional at first glance because their ORF
was blocked upstream of the DBD. In cotransfection experiments,
however, they proved to be potent inhibitors of PKA-stimulated activity
of a reporter gene construct driven by a consensus CRE. This prompted
us to investigate whether C-terminal peptides were synthesized from
these cDNAs. To allow immunological detection of such peptides, we
tagged the cDNAs by inserting the FLAG sequence between DBD I and the
3'-UTR. Using these constructs we were able to demonstrate that, 1)
C-terminal proteins were translated in vitro from CREM-17,
-20, -23, and -24 templates by internal translation initiation
downstream of the in-frame stop codons, and that 2) these proteins
bound to a CRE as homo- and heterodimers.
The methionine residues predicted to be used as start sites for
downstream translation initiation in the above constructs are Met 3
(adjacent to the P box), Met 4 in Q2, and Met 6 in module H, 19 amino
acids upstream of the DBD I. All three of these residues are also
present in CREM-2
(clone CREM-10), and Met 3 and Met
6 are present in CREM-
(our clone CREM-22), both transcript isoforms
possessing a full-length ORF. We assessed whether internal translation
initiation would also take place on these templates. By in
vitro transcription/translation, SDS-PAGE analysis, and EMSA, we
could show that shorter carboxy-terminal peptides were synthesized from
CREM-10 and CREM-22 in addition to the full-length proteins and that
long and short isoforms were cotranslated at comparable levels. A
corresponding observation has been made for the CREM-
transcript of
the mouse brain, where translation can also initiate at Met 3 to yield
the short repressor S-CREM (25). S-CREM, however, differs from
S-CREM-
2
described by us in that it includes the
second rather than the first DBD. We have thus identified four novel
CREM protein isoforms, S-CREM-
2
(20 kDa),
SS-CREM-
2
(16 kDa), S-CREM-
(13 kDa), and
SS-CREM-
(9 kDa).
We have demonstrated that these novel short isoforms are produced
in vivo and act as transcriptional repressors: 1) Expression
vectors for CREM-23, CREM-24, and CREM-17 cDNAs served as powerful
inhibitors of PKA-stimulated promoter activity; due to the upstream
in-frame stop codons, SS-CREM-2
, S-CREM-
, and
SS-CREM-
are the only carboxy-terminal products that can be
translated from these templates. 2) Protein translated from these
templates in vivo was efficiently translocated to the
nucleus. 3) The AUG codons Met 3, 4, and 6, allowing translational
initiation in vitro, are also used in vivo,
giving rise to all predicted isoforms as visualized by Western
analysis. These data indicate that CREM transcripts are polycistronic
and that short inhibitory isoforms are translated from CREM-10, -17,
-20, -22, and -24 in vivo. We were not able to identify
protein translated from CREM-23 in COS-7 cells by immunoblotting.
However, transfected CREM-23 cDNA resulted in transcriptional
repression in primary ES and in SKUT-1B cells, and C-terminal
FLAG-tagged protein appeared in the nucleus, indicating that CREM-23 is
expressed either in the form of SS-CREM-
2
or of
SS-CREM-
, at least in these uterine cell types.
The start codon for production of the shortest isoform, SS-CREM-, is
most likely Met 6, although we have no final proof for this assumption.
The other two methionine codons (Met 5 and 7) in module H are in an
unfavorable context whereas Met 6 has a purine residue at position -3
and a guanosine at +4 (GTG ATG G). This region is almost
identical to the corresponding site in exon H of the CREB
gene (GTT ATG G), which has recently been described to give
rise to the short transcriptional repressor I-CREB(s). This protein of
about 8 kDa also consists of the DBD only and is expressed in human,
mouse, and rat testis (38, 39). The nuclear translocation signal of
CREB has been defined in the first half of the DBD (RRKKKEYVK) (40).
This amino acid sequence is conserved in DBD I of CREM and is located
downstream of the last methionine residue. Therefore, even in the
unlikely case of translation initiation at Met 7, rather than Met 6,
the resultant CREM protein would contain this signal sequence.
The AUG codon in module H (Met 6) is common to all CREM transcript
isoforms. It is most efficiently used for translational initiation in
CREM-17 where no alternative more upstream AUG start codon leads to
elongational occlusion by ribosomes initiating upstream. It has been
shown that the internal AUG start codon in exon H of CREB mRNA, which
is embedded in a very similar sequence as Met 6 and gives rise to the
short repressor I-CREB(s) (8 kDa), serves as an internal ribosomal
entry site (39). Albeit less efficiently, Met 6 is also used in the
other CREM cDNAs as demonstrated by Western analysis of COS-7 cells
transfected with CREM-10, -20, -22, and -24. The SS-CREM- isoform
therefore appears to be a translational byproduct of CREM transcripts
in general. The presence of CREM transcripts in vivo,
whether or not they include transactivation domains, is therefore
expected to lead to an accumulation of SS-CREM-
repressor
protein.
Our findings not only demonstrate that one transcript can encode
several proteins (CREM-10 yields CREM-2
,
S-CREM-
2
, SS-CREM-
2
; CREM-22 yields
CREM-
and S-CREM-
), but also, conversely, that one protein can be
encoded by numerous transcripts, e.g.
SS-CREM-
2
by CREM-10 and CREM-20, S-CREM-
by
CREM-22 and CREM-24, and SS-CREM-
potentially by every CREM
transcript isoform.
The expression of a CREM transcript similar to CREM-17 has been
reported in rat spermatids. This form, CREMC-G, also lacks both
transactivation domains and the P box and includes DBD I (21). It
contains, however, the small exon X (also termed exon
), which adds
36 nucleotides 5' to the DBD I. In striking contrast to CREM-17,
however, CREM
C-G displays a full-length ORF and encodes a protein of
about 17 kDa. This is due to the insertion of one additional guanosine
in the splice junction between the first protein-coding exon (module C,
see Figs. 1
and 3
) and exon X (exon
, see Figs. 1
and 3
), which
restores the ORF leading into the DBD. As depicted in Fig. 4
, regular
splicing of module C to exons downstream of the P box leads to
disruption of the ORF. Interestingly, the junction between modules C
and H in CREM-17 is also irregular; it does not, however, restore the
ORF. Two highly related transcripts, rat isoform CREM
C-G and human
isoform CREM-17 (CREM-
1,P, 2), therefore encode dissimilar proteins
of 17- and 9-kDa molecular mass, respectively. Functionally, these
isoforms are analogs, both acting as transcriptional repressors.
We were not able to detect the upstream transactivation domain, Q1, in CREM transcripts amplified from human ES cells. Likewise, others have reported the lack of Q1 sequence in transcripts from human sources, and the sequence of the human Q1 segment has only been deduced by genomic cloning of this region using a mouse Q1 probe (41). It is uncertain whether Q1 sequence of the human CREM gene is expressed at all. Species-specific splicing has been reported for exon Z of the human CREB gene, an exon that is not spliced into rat and mouse transcripts due to mutations in the flanking splice signals (38).
Our data demonstrate that expression of the human CREM gene at the posttranscriptional level is highly complex and gains versatility both by alternative splicing and by alternative translation initiation. The majority of CREM protein isoforms represent transcriptional repressors, some of which are remarkably small.
Human ES Cells Override the Presence of Multiple Inhibitory CREM
Isoforms and of Up-Regulated ICER in the Course of Decidualization
Human ES cells decidualize in response to a sustained cAMP
stimulus. In the course of decidualization, the regulatory subunit
isoform of PKA, RI, is significantly decreased at the protein level,
while all other regulatory and catalytic subunits remain unaltered.
This leads as a net effect to an increase in free catalytic subunit,
which can traffic to the nucleus and phosphorylate target proteins such
as CREB and CREM (17). A marker of decidualization is the activation of
the dPRL promoter. This promoter is induced by cAMP via an indirect
mechanism. While induction of promoter constructs with a classical CRE
becomes detectable in transfected ES cells within 4 h of cAMP
stimulation, induction of the dPRL promoter appears with a delay of at
least 20 h. This induction can be abolished by the transfection of
an expression vector for protein kinase inhibitor (17, 42). It was of
interest to us, therefore, to investigate the expression of transducers
of the PKA-signaling pathway in ES cells undergoing decidualization.
Amplification of CREB mRNA using primers anchored in exons B and H,
upstream of the DBD sequence, yielded a single product of the predicted
size for CREB-327 (28), indicating that no alternative splicing
involving exons W or Z takes place in ES cells. These exons have been
described in human testis; their inclusion in CREB transcripts
introduces in-frame stop codons upstream of the DBD and leads to the
generation of carboxy-terminal repressors I-CREB(l) and I-CREB(s)
due to alternative translation initiation (38). No difference in the
level of CREB expression was detected when we compared undifferentiated
to differentiated ES cells. A constant level of CREB-327 is maintained
in the cells, which will be activated by phosphorylation in response to
a suitable stimulus, while no inhibitory CREB isoforms are present or
induced.
In contrast, we found a variety of novel CREM isoforms in ES cells.
While we have shown that these mRNAs are polycistronic and encode small
repressor proteins, we have not been able to investigate to what extent
these S-CREM and SS-CREM proteins are present in ES cells. We provide
evidence, however, that the downstream AUG codons are used for
translation initiation in ES cells, because transfection of
polycistronic constructs had an antagonistic effect on PKA-mediated
promoter activation. In addition, transfection of the
CREM-2
expression vector pRc/CREM-10 led to either an
enhancement or an inhibition of PKA-stimulated promoter activity
depending on the promoter/reporter construct. This indicates that not
only full-length activator protein CREM-
2
, but also
shorter repressor forms (S-CREM-
2
and/or
SS-CREM-
2
) are translated from this template. It will
be of great interest to determine whether the ratio between translation
of activator and repressor forms is hormonally regulated and linked to
the differentiation status of the cells. In general, ES cells seem to
contain more CREM transcripts potentially encoding repressors than CREM
transcripts encoding activators. The abundance of all these transcripts
does not change in the course of decidualization, but it is unknown
whether expression is regulated at the level of translation.
In contrast to the unaltered levels of CREM transcripts, we observed a dramatic up-regulation of ICER transcripts in decidualized cells. This up-regulation was dependent upon the presence of RLX in the medium and did not occur in the presence of MPA alone. Even though the RLX receptor has not been cloned to date and its signal transduction pathway has not been fully elucidated, RLX action is clearly linked to the PKA system in ES cells. Not only does RLX cause an enormous acute increase in intracellular cAMP in cultured human ES cells (43), but we have also shown that the elevated cAMP levels are maintained for at least 6 days with continued presence of RLX (17). We observed up-regulation of ICER in three independent ES cell preparations that had been induced to decidualize and activate the dPRL promoter by RLX treatment for 12, 16, or 28 days. The internal P2 promoter of the CREM gene that gives rise to ICER is cAMP-responsive. The induction of ICER has been described as transient and cell-specific. Expression in neuroendocrine cells peaks after 24 h and drops 46 h later. This is due to an autoregulatory negative feedback loop by which newly synthesized ICER protein antagonizes PKA-induced activation of the CREs in its own promoter (27). Fluctuating expression of ICER in the pineal gland in response to adrenergic signals plays an important role in circadian rhythmicity (26). In addition to neuroendocrine cells, cAMP inducibility of ICER has also been reported in human medullary thymocytes, peripheral blood T lymphocytes, and T cell lines. Again, ICER expression peaked within 3 h of stimulation (by forskolin or PGE2) and ceased soon after (44). The authors noted a relatively high level of ICER in untreated freshly prepared peripheral blood T cells, which they attributed to either an activation of cAMP signaling during preparation of the cells or to a normal physiological property of this cell type. We also observed an unexpectedly high level of ICER transcripts in decidualized ES cells treated with RLX+MPA. This was not due to the preparation procedure because 1) the cells were harvested directly into denaturing solution and 2) no up-regulation occurred in cultures harvested in parallel that had been maintained without hormone or in the presence of MPA alone. Elevated ICER was therefore caused by the long-term exposure to RLX.
Our finding of high ICER expression together with induction of the dPRL promoter in decidualized cells subjected to a persistent cAMP stimulus exerted by RLX seems paradoxical at first glance: In cotransfection experiments ICER expression vectors inhibited PKA-stimulated activity of the dPRL promoter construct dPRL-3000/luc. With regard to the cotransfection experiments, it must be remembered, however, that ICER was overexpressed in undifferentiated cells without being complemented or opposed by as yet unknown mechanisms that may evolve in differentiating cells.
What we see in decidualized cells is the apparently paradoxical
activity of two promoters in the presence of ICER, even though these
promoters are known to be repressed by isolated ICER: the dPRL promoter
and the ICER promoter itself. We suggest the following explanation for
this phenomenon: in decidualized ES cells, the repressor ICER is
titrated out by an excess of activator protein, such as
CREM-2
or CREB. Within this concept, the presence of
elevated ICER levels in cells constantly challenged by elevated cAMP
might actually be viewed as an indicator for the presence of overriding
amounts of transactivating CREM isoforms or CREB, phosphorylated by the
elevated free catalytic subunits of PKA, which in turn contribute to
the activation of decidualization markers such as the dPRL gene.
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MATERIALS AND METHODS |
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B (pos. -16 to +9; 5'-GACAAATCCAAGACAAATGA-CCATG-3'),
C (pos. 88110; 5'-ACTGGGCAAATTTCAATCCCTGC-3'),
D (pos. 121143; 5'-TGCAGTGAGCTGAGATCAGGCAC-3'). Antisense oligonucleotides were:
D' (pos. 177201; 5'-ATGTATAGTTTGGCCCGAAGGT-AAC-3'),
E (pos. 296318; 5'-AATTACACCTTCTGATTCTGCAG-3'),
F (pos. 537561; 5'-ACCATCAGATCCTGGGTTAGAA-ATC-3'),
H (pos. 765789; 5'-CAAACTTCCGGGCGATGCAGCC-ATC-3'),
J (pos. 13981423; 5'-CAGTTCATAGTTAAATATTTCTA-GTA-3'),
G (5'-GACTTGGGGCAAGTTCAGTTTCC-3'). Primer A is specific to the 5'-UTR of the ICER mRNA (45):
A (5'-TGGAACACTTTATGTTGAACTGTGG-3') Additional primers were
designed to the novel exon sequence:
' (5'-CTATGAAAATCATCAGAGACCTAGC-3') (sense) and
" (5'-CTCAATCCATCAGCAGAACAG-3') (antisense).
CREB primers were chosen upstream of the DBD of the human CREB cDNA (28):
CREB-1 (pos. 180203 in exon B; 5'-GAAGCTGAAAACCAACAAATGACA-3', sense), CREB-2 (pos. 848870 in exon H; 5'-CAATAGTGCTAGTGGGTGCTGTG-3', antisense), CREB-3 (pos. 406427 in exon C; 5'-GTGAAGATTCACAGGAGTCAGT-3', sense).
Primers for amplification of the dPRL transcript were: 98 (pos. 4372 in the decidual-specific exon 1a) (15), 173 (antisense to pos. 6787 in exon 2 of the hPRL cDNA), and 630 (antisense to pos. 710739 in the 3'-UTR) (46).
PCR on cDNA templates was performed with Taq polymerase (Promega, Madison, WI). Products were subjected to Southern blotting, hybridized with biotinylated oligonucleotide primers, and visualized with the Southern light detection system (Tropix, Bedford, MA). Genomic PCR was performed with the Expand long template PCR system on human genomic DNA (Boehringer, Mannheim, Germany). For sequencing and subcloning purposes, PCR products were isolated from a low melting agarose gel and ligated into the pGEM-T vector (Promega).
Constructs
To generate eukaryotic expression vectors, full-length CREM and
ICER cDNAs were excised from pGEM-T by ApaI digestion,
followed by polishing and NotI digestion. These fragments
with a 5'-blunt and a 3'-NotI end were inserted into pRc/CMV
(InVitrogen, San Diego, CA), which had been linearized with
HindIII, polished, and further digested with
NotI. Resultant plasmids were: pRc/CREM-10, pRc/CREM-17,
pRc/CREM-20, pRc/CREM-23, pRc/CREM-24, pRc/ICER-I, and pRc/ICER-II. For
immunological detection of CREM translation products, the FLAG sequence
(DYKDDDDK) (47) was introduced 3' to the protein-coding region,
replacing the stop codon after DBD I. CREM/FLAG fusion cDNAs were
generated by PCR on the CREM cDNAs in pGEM-T with Pfu
polymerase (Stratagene, Heidelberg, Germany) using oligonucleotide B
(see above) as upstream primer. The following antisense oligonucleotide
served as the downstream primer: 5'-GAATTGCGGCCGC TTA
TTT GTC GTC ATC GTC TTT GTA GTC CTC TAC TTT ATG GCA ATA-3'; it
contained a NotI site (underlined), the FLAG
sequence terminated by a stop codon (in bold type), and
CREM-specific sequence (pos. 979996 in DBD I). After NotI
digestion, the PCR products had a 5'-blunt and a 3'-NotI end
and were inserted into pRc/CMV, which had been treated as described
above to have a 5'-blunt and a 3'-NotI end. Thus,
CREM-10/FLAG, CREM-17/FLAG, CREM-20/FLAG, CREM-22/FLAG, CREM-23/FLAG,
and CREM-24/FLAG were created.
The expression vector for the catalytic subunit of PKA, pRSV-Cß, and an inactive mutant thereof, pRSV-Cßmutant, were kindly provided by Dr. R. Maurer (Oregon Health Sciences University, Portland, OR) (48). The generation of a reporter gene construct in pGL2-Basic (Promega) carrying 3000 bp of DNA flanking the dPRL promoter (dPRL-3000/luc) has been described previously (16). A fragment truncated to -332 bp was generated by PCR and inserted into pGL3-Basic (Promega) to yield dPRL-332/luc3. Plasmid CRE/-36rPRL/luc (kindly provided by Dr. M. G. Rosenfeld, Howard Hughes Medical Institute, San Diego, CA) (49) contains a composite CRE element 5'-TTGGCTGACGTCAGAGAGAGGCCGGCCCCTTACGTCAGAGGCGAG-3' linked to the minimal rPRL promoter fragment -36/+34. This fusion was isolated by PCR with the upstream primer 5'-CTTGG-CTGACGTCAGAGAGAG-3' and the downstream GL2 primer (Promega) antisense to the luciferase reporter gene. The product was restricted with HindIII at the 3'-end of the rPRL sequence and inserted into SmaI/HindIII-digested pGL3-Basic to give pCRE/-36rPRL/luc3.
Cell Culture
Purified cultures of human ES cells were prepared as previously
described (16) and plated in basal media, a 1:1 mixture of DMEM and
Hams F-12 containing 10% FCS that had been depleted of steroids by
treatment with dextran-coated charcoal, 100 U/ml penicillin, and 100
µg/ml streptomycin, and supplemented with 10-9
M 17ß-estradiol and 1 µg/ml insulin. Cells kept under
these conditions were referred to as -/- cells. Optionally basal
medium was further supplemented with 2.5 x 10-7
M MPA (Sigma) (+/- cells) or with MPA and 100 ng/ml
porcine relaxin (3000 U/mg protein; kindly supplied by S. Raiti,
National Hormone and Pituitary Program, NIDDK, Baltimore, MD) (+/+
cells). Media were changed every 23 days. When they had reached
confluence, monolayers were subcultured at a ratio of 1:3.
The human uterine sarcoma cell line SKUT-1B (HTB 115, American Type Culture Collection, Rockville, MD), obtained from the European Collection of Animal Cell Cultures (Salisbury, UK), was maintained in DMEM/Hams F-12 (1:1) with 10% FCS, 50 U/ml penicillin, and 50 µg/ml streptomycin. COS-7 cells were cultured in DMEM with 10% FCS and antibiotics as above.
Transient Transfections, Nuclear Extract Preparations, and
DNA-Binding Studies
Transfections were performed by the calcium phosphate
precipitation method on SKUT-1B cells and on ES cells of the first
passage plated in 12-well plates. SKUT-1B cells received 1.4 µg
reporter plasmid, 0.6 µg pRSV-Cß (or pRSV-Cßmutant
for controls), and 0.8 µg pRc/CREM or pRc/ICER constructs. For ES
cells, 0.8 µg pRSV-Cß was used. The DNA precipitate was removed
from the cells 16 h later and replaced by fresh medium, and cells
were harvested after an additional 24 h for luciferase assay
(Promega). All transfections were performed in triplicate wells on
several independent cell preparations.
For preparation of nuclear extracts from COS-7 cells, transfections were performed in 6-cm dishes with a mixture of 5.5 µg CREM/FLAG constructs and 30 µl DOTAP (Boehringer) made up to a total volume of 150 µl with HEPES-buffered saline. The DNA/DOTAP mixture was added to the culture medium and left on the cells for 16 h. Medium was then replaced, and cells were harvested 24 h later in buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM dithiothreitol, and protease inhibitor cocktail (Complete; Boehringer)] and allowed to swell on ice for 5 min, after which NP-40 was added to 1%, and nuclei were spun down. The nuclear pellet was resuspended in buffer B (same as buffer A including 400 mM NaCl and 1% NP-40), and nuclear proteins were extracted for 15 min at 4 C.
EMSAs were performed using 10 µg nuclear extracts, 3 µl in vitro translation product, or 0.3 µg human CREB-1 bZIP domain (amino acids 254327; Santa Cruz Biotechnology, Santa Cruz, CA). The CRE probe was a 32P-end-labeled double-stranded oligonucleotide with a consensus CRE (5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'). Protein was incubated in bandshift buffer (40 mM HEPES, pH 7.9, 20 mM MgCl2, 240 mM KCl, 5 mM spermidine, 16% Ficoll) with 1 µg poly(deoxyinosinic acid-deoxycytidylic acid) for 15 min (competition was carried out in the presence of 100-fold molar excess of unlabeled CRE), followed by a 45-min incubation with probe (20,000 cpm) on ice. For supershift studies, incubation with probe was reduced to 30 min on ice, followed by addition of 1 µl FLAG antiserum and a 20-min incubation at room temperature. Reactions were then resolved on 6% polyacrylamide gels in Tris-borate-EDTA buffer.
Protein Analysis
CREM/FLAG constructs (0.5 µg) were subjected to in
vitro transcription and translation using the TNT T7 coupled
reticulocyte lysate transcription/translation system (Promega) in the
presence of [35S]methionine in a final volume of 25 µl.
Of this reaction, 20 µl were immunoprecipitated by incubation with
FLAG antiserum (D-8; Santa Cruz Biotechnology) followed by protein A
Sepharose (Pharmacia, Uppsala, Sweden) in Ripa buffer [PBS, pH 7.4,
1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor set
(Boehringer)]. Products were resolved on 327% or 1020%
polyacrylamide SDS gels (ICN, Meckenheim, Germany) and
autoradiographed. For use of proteins in DNA-binding studies, in
vitro transcription/translation was performed with unlabeled
methionine.
Nuclear extracts from COS-7 cells transfected with CREM/FLAG constructs were immunoprecipitated with FLAG antiserum D-8 as above and fractionated on a 16.5% SDS polyacrylamide gel (50). Proteins were transferred to polyvinylidene fluoride membrane (Immobilon P; Millipore, Eschborn, Germany) in 0.025 M Tris, 0.192 M glycine, 20% methanol at 250 mA for 3 h. Immunodecoration was performed with FLAG antiserum D-8 followed by detection with the enhanced chemiluminescence (ECL) system (Amersham, Braunschweig, Germany) or with antibody against human CREM-1 (Santa Cruz) and the Western Light chemiluminescent detection kit (Tropix, Bedford, MA).
Immunofluorescence
SKUT-1B cells were plated in 24-well plates coated with
poly-D-lysine (1 µg/cm2; Sigma Chemical Co.,
St. Louis, MO) and transiently transfected with 1 µg CREM/FLAG
constructs as described above. Medium was replaced 16 h later, and
cells were incubated for an additional 24 h. Cells were then
washed in PBS, fixed in 2.5% paraformaldehyde in PBS for 10 min at 37
C, washed in PBS, and permeabilized with 0.2% Triton X-100 in PBS for
10 min. To block nonspecific binding, 200 µl normal goat serum
(Sigma) were added, left on the wells for 5 min and aspirated without
subsequent wash. Monoclonal FLAG antibody M2 (Eastman Kodak, New Haven,
CT) (20 µg/ml in PBS) or FLAG antiserum D-8 (Santa Cruz) (0.2 µg/ml
in PBS) was added for 1 h at room temperature, followed by three
washes in 0.001 M PBS. Fluorochrome-conjugated secondary
antibody (anti-mouse Cy3 affinipure F(ab')2 goat IgG, or
anti-rabbit Cy3 affinipure goat IgG; Dianova, Hamburg, Germany) was
diluted 1:100 in 0.001 M PBS/2% normal goat serum and
applied for 60 min, followed by three washes in 0.001 M
PBS. Stained cells were visualized on an inverted microscope at
510560 nm.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was presented in part at the 10th International Congress of Endocrinology, San Francisco, CA, June 1215, 1996.
This work is based in part on the doctoral study by R. T. performed at the Faculty of Biology, University of Hamburg, and was supported by Deutsche Forschungsgemeinschaft Grant Ge 748/1-2.
Received for publication August 26, 1996. Revision received October 21, 1996. Accepted for publication October 24, 1996.
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REFERENCES |
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