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Isoforms Expressed by Two Newly Identified cAMP-Responsive Promoters Active in the Testis1
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Joel F. Habener, M.D., Laboratory of Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit Street, WEL320, Boston, Massachusetts 02114. E-mail: jhabener{at}partners.org
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Abstract |
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) and
repressor (
) forms of CREM, whereas the cAMP-responsive P2 promoter
activates the formation of messenger RNAs encoding the inducible cAMP
early repressor. Here we report the identification of two additional
promoters in the CREM gene, P3 and P4, that in the rat testis encode
two novel transcriptional activator CREM isoforms, termed CREM 
1 and
CREM 2, respectively. Notably, the P3 and P4 promoters are activated
by cAMP-dependent protein kinase, thereby providing cAMP-regulated
transcription of CREM activators in addition to the established
cAMP-regulated inducible cAMP early repressor. Analysis ex
vivo of CREM gene expression in temporally staged segments of
the seminiferous tubule during the spermatogenic cycle shows that the
activities of the P1, P3, and P4 promoters are independently regulated.
Our identification of the cAMP-activated P3 and P4 promoters that
direct expression of the novel 1 and 2 activator isoforms of CREM
brings further insight into the complex expression of the CREM gene
during germ cell development and may have implications in understanding
the control of fertility. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CREM gene consists of 10 exons, several of which are alternatively
spliced in the formation of messenger RNAs (mRNAs; Fig. 1
, A and B). The upstream promoter of the
CREM gene (P1) is believed to be constitutively active. The central
region of CREM is encoded by exons E and F that contain a protein
kinase A phosphorylation site (3). When phosphorylated,
this kinase-inducible domain (KID) interacts with coactivators such as
CBP/P300 and thereby mediates cAMP inducibility. Two glutamine-rich
trans-activation domains, termed domains, flank the KID
domain. The upstream domain (1) is coded for by exon C and the
3'-region of exon B. The downstream domain (2) is encoded by exon G.
The DNA-binding and dimerization domain, a basic region-leucine zipper
(bZIP) structure, is encoded by exons H and I. However, exon I, which
includes the 3'-untranslated region (UTR), has two alternative splice
sites that give rise to alternative DNA-binding domains (DBD1 and
DBD2).
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trans-activation domains. Furthermore,
an alternative promoter, P2, produces a short form of the mRNA
including the bZIP domain, but not the KID or the
trans-activation domains (5). The P2 promoter
contains four CREs (termed CAREs) and is stimulated strongly by cAMP
(6, 7, 8, 9). The protein encoded by the mRNA transcribed from
the P2 promoter is termed the inducible cAMP early repressor (ICER),
and represses cAMP-regulated gene expression (7). The
activity of the P1 promoter has not been fully characterized, but is
believed to direct constitutive tissue-specific expression
(1, 10).
In germ cells of the testis large amounts of mRNAs for the activator
forms of CREM (CREM , 1, and 2) are produced
(11). The mRNAs for the isoforms are detectable in
premeiotic pachytene spermatocytes, but the proteins are detected only
in postmeiotic round spermatids, probably as a result of mRNA
sequestration and delayed translation (3, 11). The factors
directing the preferential splicing and accumulation of CREM mRNA
are unknown; however, it has been shown that an accompanying shift of
polyadenylation sites to ones closer to the 3'-end of the protein
reading frame contributes to increased mRNA stability
(12). CREM is required for the completion of germ cell
development because crem null mice display an arrest in spermatogenesis
at the early haploid phase and fail to differentiate round spermatids
to spermatozoa (13, 14). Notably, in the germ cells CREM
is coexpressed with the coactivator, activator of CREM in the
testis (ACT). ACT does not require the phosphorylation of KID for
association with CREM or trans-activation, effectively
making CREM a constitutive activator (15). Herein, we
report the identification of two new CREM promoters, P3 and P4, active
in the testis and giving rise to two novel activator isoforms of CREM,
CREM 1 and CREM 2. Both the P3 and P4 promoters are cAMP
responsive, indicating that cAMP signaling activates the expression of
trans-activator forms of CREM as well as the repressor ICER
CREM isoform. Further, we show that the activation of the P1, P3, and
P4 promoters occurs at different temporal stages of the cycle of the
seminiferous epithelium in the rat. Our findings provide further
evidence suggesting that CREM gene expression may be important in the
testis and in the control of spermatogenesis.
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Materials and Methods |
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PCR products were prepared for TA cloning into pCR2.1-TOPO (Invitrogen, Carlsbad, CA) by phenol/chloroform extraction and ethanol precipitation, followed by incubation with Taq polymerase at 72 C for 10 min in the presence of 1 mM deoxy-ATP.
Database searches
Homology searches of the DBEST database were performed with
BLAST software accessed through the NCBI web page
(www.ncbi.nlm.nih.gov). The working draft sequence from human
chromosome 10 (unpublished direct submission by S. Sims, Sanger Center,
Hinxton, UK) is available through GenBank under accession number
AL157783.
PCR amplification from genomic DNA
Amplification from rat genomic DNA was carried out using the
TaqPlus long PCR system (Stratagene). The 6.5-kb fragment
between exon B and exon 1 was amplified in high salt buffer, using a
nested PCR method. The first PCR amplification was a touchdown protocol
of 10-sec denaturation (94 C) followed by 5-min annealing and
extension. The annealing/extension temperature was set at 72 C for the
first 5 cycles, 70 C for the next 5 cycles, and 68 C for 30 cycles with
5-min extension time. The primers were: forward, RCRMF61
(5'-catagctccttgcatatgcagactgg-3'); and reverse, CRMVR1
(5'-cttcttctacaggatgcctgaagc-3'). The second round of PCR amplification
used conditions of 94 C for 4 sec, 58 C for 10 sec, and 72 C for 5 min
for 30 cycles and the following primers: forward, RCRMF87
(5'-ccaaatttctgtccctactctagc-3'); and reverse, CRMVR2
(5'-taggttatgctgatgccacc-3'). The 6.5-kb fragment between exon 1 and
exon C was amplified by nested PCR with first round conditions of 94 C
for 4 sec and 68 C for 5 min for 30 rounds. The primers were: forward,
RCP3F (5'-ggctatcaatcgcatcaccttacc-3'); and reverse, RCRMR206
(5'-actccctggacctgtacagtttgg-3'). In the second round, conditions were
94 C for 4 sec, 58 C for 10 sec, and 72 C for 5 min for 30 cycles. The
primers were: forward, CRMVF1 (5'-ttagtgggttttcagtggatgtgg-3'); and
reverse, RCRMR170 (5'-tgtactagagtcacagctgg-3'). PCR products were
cloned into pCR2.1-TOPO as described above.
cDNA synthesis and RT-PCR analysis
Isolation of total RNA and synthesis of cDNA was carried out as
described previously (16). cDNA samples were prepared from
seminiferous tubule segments staged by transillumination and pooled for
RNA extraction and cDNA synthesis (16, 17).
PCR detection of distinct CREM isoforms was carried out with forward
primers specific for exons B (CRMF87; see above), 1 (CRMVF1; see
above), and 2 (MCP4F1, 5'-ccaggacagtgactacctcc-3'). Reverse primers
were specific for exon Ia (CREMR5, 5'-ccaattcacactctacagcag-3') and
exon Ib (CREMIb2, 5'-aatatttctactaatctgttttgggagagc-3'). PCR conditions
in all reactions were 94 C for 4 sec, 58 C for 10 sec, and 72 C 1 min
for 30 cycles. PCR reactions were performed in 30-µl reactions using
1.2 µl of templates. Reactions contained 20 pmol each of forward and
reverse primers, 0.2 mM each of deoxy-NTPs, and 1.5 U
thermostable Taq polymerase (TaKaRa Biomedical, Inc.,
Berkeley, CA). Where PCR products were cloned, 50-µl reactions using
2 µl template were used, and high fidelity Pfu polymerase
was substituted. Products were ligated into pCR2.1-TOPO after being
prepared as described above.
The identities of CREM isoforms were confirmed by hybridization with
probes for exon C/1 (RCRMR143, 5'-gatctcgag cttccagtgcctgatccagc-3')
and exon G/2 (CREMF507, 5'-agcccaaggtggaacaatcc-3'). Hybridization
with [
-32P]ATP-labeled oligonucleotide
probes was performed in a solution of 5 x SSC (standard saline
citrate), 1% SDS, 10 x Denhardt’s solution, and 100 µg/ml
denatured salmon sperm DNA for 3 h at 37 C. Blots were washed to a
maximum stringency of 0.5 x SSC at 52 C. For rehybridization, the
labeled probe was removed by washing blots in 0.5 M NaOH at
room temperature for 1 h, then rinsing in 2 x SSC.
Construction of reporter plasmids
Reporter constructs pRCP3(-300)luc and pRCP3(-564)luc were
developed using Pfu polymerase PCR products generated from
the cloned rat genomic DNA fragment encompassing the P3 promoter. The
primers were: forward, RCPF300
(5'-ctggagctcctatattgtgaattcacatttctctaatcc-3') and RCPF564
(5'-ctggagctccccaaatgctgctgggattaaagg-3'); and reverse, RCPR1
(5'-gatctcgagcactaaagacagttgttaatctgaagaagc-3'). These fragments were
subcloned directionally into the luciferase gene-containing plasmid
pGL3-Basic (Promega Corp., Madison, WI) using the
XhoI and SacI sites.
Reporter construct pRCP4(-296)luc was developed in a similar manner using the cloned rat genomic DNA fragment encompassing the P4 promoter and the following primers: forward, RCP4F297 (5'-gatgagctcaaatagtgaaagaattgccgtatgc-3'); and reverse, MCP4R1 (5'-gatctcgagggaggtagtcactgtcctgg-3'). For pRCP4(-296+Int)luc, the reverse primer was RCRMR143 (see above). Mutation of the ATG codon (translational start codon) was achieved with primers RCP4M1F1 (5'-ggacagtgactacctcctaaaggtaagtaccc-3') and RCP4M1R1 (5'-gggtacttacctttaggaggtagtcactgtcc-3') using methods previously described (18).
The rat ICER promoter construct pRCP2(-308)luc was constructed from pGL3-Basic using a 308-bp segment of rat genomic DNA amplified with primers based on the mouse sequence (5), ICERPF1 (5'-gatttttgttcagtccctgaaatgtgg-3') and ICERPR1 (5'-gttgggcttttgcatatagagtgg-3'), using conditions of 94 C for 4 sec, 55 C for 10 sec, and 72 C for 30 sec for 35 cycles.
Cell transfection and gene expression studies
Promoter regulation by protein kinase A (PKA) was studied by
transient transfection in placental JEG-3 cells (American Type Culture Collection, Manassas, VA; HTB-36). Transfections were
carried out in 24-well plates in duplicate, in 0.5 ml OptiMEM
(Life Technologies, Inc., Gaithersburg, MD)/well, using 2
µl Lipofectamine (Life Technologies, Inc.), 300 ng
reporter, and 100 ng PKA catalytic subunit expression vector or control
plasmid pcDNA3.1 (Invitrogen). Luciferase assays were carried out
18–24 h posttransfection using the Promega Corp.
luciferase assay system (Madison, WI).
Comparisons of pRCP4(-296)luc and pRCP4(-296-Int)luc were carried out in primary rat spermatocytes by a modification of the procedure described previously (17). Partially purified spermatocytes were isolated from the testes of 24- to 30-day-old Sprague Dawley rats by sequential digestion with collagenase and trypsin, then plated for 4 h in DMEM and FCS. Nonadherent cells were harvested, washed, and replated in serum-free OptiMEM in 12-well plates (3 x 105 cells/well). Transfection was carried out with 5 µl Genefector (Venn-Nova, Pompano Beach, FL), and 500 ng reporter construct/well. Transfections were carried out in duplicate. Luciferase assays were performed as described above. All animal protocols used in this study were subject to review and were approved by an animal ethics committee.
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1 domain. The novel region of
DNA also includes 60 nucleotides of putative 5'UTR (Fig. 1C
). Using PCR
from rat genomic DNA, the novel CREM sequence was mapped to a site
approximately 6.5 kb downstream from exon B and 6.5 kb upstream from
exon C (Fig. 1E). Sequencing of these genomic DNA PCR products
established that the new 5'-region represents a single exon, hereby
termed exon 1.
To confirm the existence of the novel 5'-sequence, a series of homology
searches was performed against the DBEST database, a repository of
partial cDNA sequences from cDNA libraries, termed expressed sequence
tags (ESTs). A search using the previously described mouse CREM
sequence up to the end of exon B returned five cloned sequences from
placenta, brain, mammary, and thymus, confirming the widespread
expression of the CREM isoforms. Searching with the novel exon 1
sequence found two matches from mouse testis cDNA libraries (accession
no. AI386461.1 and AA145609.1). However, unlike the
5'-ligation-mediated PCR products, the 1 exon in both mouse ESTs was
spliced directly to exon E, the first exon of the extended KID
domain.
Surprisingly, a search with mouse CREM exon C revealed a second novel
5'-region present in four mouse clones from mouse testis cDNA libraries
(accession no. AI385961, AI326536, AA062413, and AA061566). Three other
clones, from brain and placenta, were found containing the previously
described first exons, A and B. The second novel 5'-sequence, termed
exon 2, is 34 bp in the longest clone and has a potential in-frame
start codon at the 3'-end (Fig. 1D). A single human clone (accession
no. T29820) with a homologous short 5'-region spliced to the KID was
also reported in the EST database, derived from a testis cDNA library.
Exon 2 was located in rat genomic DNA from the PCR product spanning
exon 1 and exon C. A working draft sequence from human chromosome 10
(accession no. AL157783) that includes exon 2 and the surrounding
sequence was also compared. Exon 2 was located close to the
5'-boundary of exon C in both rat and human genomic DNA, separated by a
short intron of 145 or 146 bp, respectively (Fig. 1E). Both of the
novel exons are distinct from another recently described CREM exon,
exon 
(19). Exons 1 and 2 represent novel
transcription start sites for CREM mRNAs in the testis. The distance
between the exons and the previously described exon A suggests that the
transcription of all three mRNAs is regulated by separate promoters.
The identification of the P3 and P4 promoters brings the total number
of CREM gene promoters to four (P1, P2, P3, and P4).
In an analysis of CREM sequences obtained from GenBank, an additional polymorphism was detected. The CREM sequence from the human genome (accession no. AL157783) differs from three human CREM cDNA sequences (accession no. S68271, S68134, and NM001881) by 13 bp at the start of exon C. The human genome sequence (GTTTCTGTGGCTG) is homologous to the equivalent rat and mouse sequences. Despite the mismatch at the start of exon C, a search of the 190-kb human working draft sequence with the previously reported human CREM cDNA sequence from the start of exon C (TGCAGTGAGCTGC) returned seven matches. When the search sequence string was shortened to the first 10 bases only, 51 matches were found. The full 13-bp sequence also occurs as part of the Alu repeat consensus motif (20). These findings suggest the existence of a polymorphism in the human CREM gene resulting from the insertion of a partial Alu element at the 5'-end of exon C.
Prevalence and expression patterns of CREM isoforms
The prevalent forms of CREM in the rat testis were assessed by
RT-PCR using forward primers specific for exons 1, 2, and B.
Reverse primers were specific for either of the two DNA-binding domains
(alternatively spliced exons Ia and Ib). Detection of the longer CREM
isoforms in cDNA prepared from tissues other than testis (hypothalamus,
cerebellum and pancreatic islets) was not reliably achieved for any of
the primer combinations, even with longer 35-cycle PCR conditions (data
not shown). Using testicular cDNA, PCR products are detected with all
three forward primers and the exon Ib reverse primer (Fig. 2A, upper
panel). All three forward primers detect two
prevalent DNA products. The identity of these products was first
determined by hybridization with oligonucleotide probes for the CREM
2 and 1 exons (Fig. 2A, middle and lower
panels, respectively) and was further confirmed by cloning and
nucleotide sequencing. The two prevalent products were identified as
transcripts containing either both exons (upper product) or only
the 2 exon (lower product) as well as the alternatively spliced
exon. The ratios of and 2 products vary significantly depending
on the first exon used. CREM 1 isoforms preferentially splice the
first exon to exon E, excluding the 1 domain, whereas CREM 2
isoforms preferentially splice to exon C. The reverse primer in this
experiment was specific for exon Ib, although equivalent products were
also detected with the exon Ia-specific primer as shown below.
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(16, 17). All combinations of forward and reverse
primers were tested. The results indicate stage-specific cycling of
CREM expression, with distinct expression profiles for the exon 1-,
2-, and B-containing isoforms. Whereas the exon B- and 1-specific
primer combinations detect maximal product in spermatogenic stages
I–VII, the 2-specific primer combinations detect maximal product in
stages IX—IV. These findings provide further evidence for the
independent regulation of the P1, P3, and P4 promoters associated with
each distinct alternative first exon. The choice of reverse primer (exon Ia or Ib) has little or no effect on the stage-specific expression profile, showing that DNA-binding domain selection is not regulated in any detectable stage-dependent manner. Interestingly, the exon Ia-specific primer always gives less product compared with the exon Ib-specific primer. Control experiments in which 5 pg of plasmids containing cloned CREM-Ia and CREM-Ib isoforms were amplified with different primer combinations failed to show any difference in priming efficiency between the Ia- and Ib-specific primers. These findings suggest that the second DNA-binding domain of CREM DBD2 is preferentially included in CREM mRNAs expressed in the testis.
The ages at which the different CREM isoforms are first detected in rat
testis were assessed using the three forward primers in combination
with the exon Ib-specific reverse primer (Fig. 2C). The mRNAs from
promoters P1 and P4 (exons B and 2, respectively) were detectable
well in advance of the P3-derived 1 mRNA. It has been previously
observed that CREM mRNA is detectable in pachytene spermatocytes
well before the appearance of the translated protein in postmeiotic
round spermatids (11, 3). Our findings indicate that this
previously transcribed CREM mRNA is actually the product of both the
P1 and P4 (CREM 2) promoters, active as early as 20–22 days in the
developing rat testis. The P3-derived mRNA (CREM 1) is not
substantially expressed until well after the emergence of round
spermatids, which occurs at or around day 25. This dichotomy in the
activation of the multiple CREM promoter activities probably reflects
premeiotic (20–22 days) and postmeiotic (25 days) activation of the
promoters.
Promoter sequence and regulation
Consensus sequences for the rat P3 and P4 promoters were
determined by analysis of PCR products generated from genomic DNA.
Initial analysis of the rat P3 promoter (Fig. 3A) revealed the presence of 6 motifs
bearing similarity (5 out of 8 bp) to the consensus cAMP response
element (5'-TGACGTCA-3') (21, 22). Two of the sites
conserve the 5 bp TGACG half-site common to many functional CREs. The
presence of potential CREs was of particular relevance given that the
levels of P3-derived mRNA are highest during the stages of the
spermatogenic cycle when cAMP production is maximal (23)
(Fig. 2B). Additionally, the CREs may be binding sites for the CREM
protein, accounting for the probable postmeiotic activation of the P3
promoter (Fig. 2C). Two reporter plasmids containing different lengths
of the P3 promoter and CREM 1 5'UTR were constructed. P3 promoter
sequence in pRCP3(-564)luc extends from positions 1 to 552 in Fig. 3A
and includes all of the potential CREs. The pRCP3(-300)luc construct
includes nucleotides 265–552 and the five proximal CRE-like
elements. In addition, pRCP2(-308), a construct based on the rat ICER
promoter (P2), was prepared using primers based on the mouse ICER
promoter (5). Analysis of the rat P2 promoter sequence
showed 81.7% conservation with the mouse, with two of the four CREs
(CAREs) completely conserved, and one partially conserved (data not
shown). Regulation by cAMP-inducible protein kinase was assessed by
cotransfection with an expression vector for PKA catalytic subunit.
Both P3 constructs showed significant induction by PKA (Fig. 3B).
Overall, the P2 promoter showed the greatest PKA-induced activity
(>2-fold over pRCP3(-564)luc); however, fold induction by PKA was
lower (34-fold) than that with P3 (131- to 164-fold) due to higher P2
basal activity (Fig. 3B).
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1 to
exon C. The rat sequence was compared with the human genomic DNA
working draft sequence. Substantial homology (60%) was evident up to
296 bp upstream of the P4 promoter (Fig. 4A), and this similarity extended through
the downstream 145/146-bp intron (59.3% homology; Fig. 4B). This high
degree of conservation of noncoding sequence between the human and rat
suggests that elements within the 2-1 intron may influence
promoter activity.
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). pRCP4(-296+Int)luc
includes 299 bp of the promoter and 5'UTR as well as the following
145-bp intron (Fig. 4B) and 32 bp of exon C. To prevent suppression of
the luciferase reporter gene, the putative start codon was mutated to
AAT. These constructs were first transfected into primary rat testis
cultures from 24- to 30-day-old rats. The prevalent cells in these
cultures were spermatocytes. As a positive control for transfection
efficiency, a construct based on 303 bp of the pituitary adenylate
cyclase-activating polypeptide testis-specific promoter (pPPT 303luc)
(17) was transfected in parallel. Both P4-based constructs
expressed luciferase activity in the transfected primary cell cultures
above that of the promoterless vector (Fig. 4B), with no significant
contribution from the 2-1 intron. However, the activity of the
pPPT303luc in these experiments was 24-fold greater (data not shown),
suggesting that the P4 constructs are providing relatively low level
promoter activity in this assay.
The P4 constructs were also tested for activation by PKA in JEG-3
cells. In these experiments, there was superactivation by cotransfected
PKA of approximately 3-fold for both constructs (Fig. 4D). However, the
presence of the 2-1 intron increased both basal and
PKA-stimulated reporter expression by a factor of approximately 6,
suggesting that this region may have a role in promoter activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2) products in rat testes as early as 22
days of age is consistent with activation of these promoters in
pachytene spermatocytes before meiosis, whereas appearance of the P3
(1) products probably coincides with the accumulation of postmeiotic
round spermatids. Furthermore, stage-specific expression profiles show
that the P1 and P4 promoters are not coregulated.
Splicing patterns of the CREM mRNAs also vary considerably. The
P1-derived mRNA isoforms seem to splice the 5'-region (exons A and B)
with equal frequency to either the 1 exon or directly to the KID
domain. Upon translation, these mRNAs would give rise to CREM or
CREM 2, respectively. The P3-derived mRNA preferentially splices the
1 exon to the KID domain, omitting the 1 exon producing CREM
1/2. The opposite pattern is seen for the P4-derived mRNA, with
the majority of the mRNA containing both exons (CREM 2/).
Western blot analysis of testicular CREM has shown that the majority of
the protein is of the isoform, containing both 1 and 2
trans-activation domains (3). Although not
entirely quantitative, analyses of the relative expression of the
different CREM mRNAs in the testis suggest that the 1 and 2 mRNAs
are significantly more abundant than the exon B-containing mRNAs. An
important consideration is whether these 1 and 2 mRNAs are
efficiently translated into CREM protein isoforms in the testis.
Analyses of CREM protein expression await the development of specific
antisera to the unique amino acid sequence of CREM 1. As for the
question of tissue distribution, it is not yet certain that the new P3
and P4 promoters are completely inactive in other tissues. The EST data
available to date draw a firm correlation between the testis and both
CREM isoforms. In total, DBEST contained two 1- containing
ESTs from mouse, four 2-containing EST’s from mouse, and one
2-containing EST from human, and all are from testis cDNA-containing
libraries. Longer CREM isoforms, such as CREM and CREM , have
been reported to be expressed in the brain (10). However,
in the brain and other tissues, CREM mRNAs are present at low levels
compared with those in the testis, and the role of longer CREM proteins
is uncertain.
The importance of CREM expression outside the testis has been
proven for the shorter ICER proteins, which are expressed under the
control of the P2 promoter (5). In the crem null mice, the
absence of the ICER protein in the pituitary intermediate lobe leads to
a dysregulation of ß-endorphin levels (24). In contrast,
no essential functions for longer forms of CREM have been demonstrated,
other than control of male germ cell development by CREM isoforms.
The crem null mice are infertile, with an arrest of spermatid
development after meiosis (13, 14). Low levels of
CREM expression have also been associated with failures in human sperm
production (25, 26).
One important observation is that the P3 and P4 promoters active in
germ cells are inducible by the cAMP/PKA/CREB pathway. The
cAMP-responsive P2 promoter is not reported to be active in male germ
cells, although it is active in Sertoli cells (6). The P3
promoter is more sensitive than the P4 promoter with regard to PKA
inducibility and contains several potential CREs. CREs are binding
sites for CREM as well as CREB (1), and the detection of
CREM 1 mRNA only in rat testes older than 25 days may be evidence of
an autopositive feedback effect of CREM on the P3 promoter in
postmeiotic germ cells.
The regulation of the P1 and P4 promoters also warrants further investigation, both for the potential of autoregulation by CREM proteins and to identify tissue-specifying elements and trans-activating factors. Initially, the increased production of CREM was hypothesized to be related to RNA stability (11). A shift in polyadenylation site brought about by FSH action was postulated to be a key regulatory event. Recent evidence from primates and rodents, however, shows that CREM production is maintained despite gonadotropin deficiency (27) and is probably a function of germ cell maturation. The existence of multiple, independently regulated promoters for CREM in the testis further raises the possibility of control at the level of transcription in addition to RNA stability.
Although no regulatory elements are firmly identified within the
conserved portions of the P4 promoter, one intriguing possibility is
the occurrence of a 5'-GGAA-3' motif at positions 90/92 (Fig. 4A). This
binding motif for Ets family transcription factors is part of an
essential element in the male germ cell-specific promoter of the murine
ß4-galactosyltransferase I gene (28), the expression of
which occurs in late pachytene spermatocytes and round spermatids. This
pattern of expression is similar to that of CREM .
A question not yet addressed is whether the CREM isoforms have
specific functions due to the alternative (CREM 1) or absent (CREM
2) amino-terminal sequence. Notably, however, the splicing of the
1 exon creates putative phosphorylation motifs for casein kinases
type I and possibly type II (EEDYSSGD) and cAMP-dependent PKA (KKVSV).
Studies to date have not suggested a specific function for the region
preceding the first glutamine-rich domain in either CREM or CREB,
although the presence of four glutamine residues in the exon B coding
sequence suggests that it may contribute to
trans-activation. Certainly, the effects of different CREM
domains on interactions with ACT, a major coactivator of CREM in the
testis, have not yet been fully examined.
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Acknowledgments |
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Footnotes |
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2 Investigator with the Howard Hughes Medical Institute.
Received June 12, 2000.
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 G. L. Christensen, S. P. Wooding, I. P. Ivanov, J. F. Atkins, and D. T. Carrell Sequencing and haplotype analysis of the Activator of CREM in the Testis (ACT) gene in populations of fertile and infertile males. Mol. Hum. Reprod., April 1, 2006; 12(4): 257 - 262. [Abstract] [Full Text] [PDF]
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K. Vouk, P. Hudler, L. Strmsnik, M. Fink, G. Majdic, B. Zorn, E. Lalli, P. Sassone-Corsi, N. Debeljak, R. Komel, et al. Combinations of genetic changes in the human cAMP-responsive element modulator gene: a clue towards understanding some forms of male infertility? Mol. Hum. Reprod., August 1, 2005; 11(8): 567 - 574. [Abstract] [Full Text] [PDF]
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 S. Kimmins, N. Kotaja, I. Davidson, and P. Sassone-Corsi Testis-specific transcription mechanisms promoting male germ-cell differentiation Reproduction, July 1, 2004; 128(1): 5 - 12. [Abstract] [Full Text] [PDF]
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K. Steger, R. Behr, I. Kleiner, G. F. Weinbauer, and M. Bergmann Expression of activator of CREM in the testis (ACT) during normal and impaired spermatogenesis: correlation with CREM expression Mol. Hum. Reprod., February 1, 2004; 10(2): 129 - 135. [Abstract] [Full Text] [PDF]
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 K. Tenbrock, Y.-T. Juang, M. Tolnay, and G. C. Tsokos The Cyclic Adenosine 5'-Monophosphate Response Element Modulator Suppresses IL-2 Production in Stimulated T Cells by a Chromatin-Dependent Mechanism J. Immunol., March 15, 2003; 170(6): 2971 - 2976. [Abstract] [Full Text] [PDF]
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B. Gellersen, R. Kempf, R. Sandhowe, G. F. Weinbauer, and R. Behr Novel leader exons of the cyclic adenosine 3',5'-monophosphate response element modulator (CREM) gene, transcribed from promoters P3 and P4, are highly testis-specific in primates Mol. Hum. Reprod., November 1, 2002; 8(11): 965 - 976. [Abstract] [Full Text] [PDF]
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K. Tenbrock, Y.-T. Juang, M. F. Gourley, M. P. Nambiar, and G. C. Tsokos Antisense Cyclic Adenosine 5'-Monophosphate Response Element Modulator Up-Regulates IL-2 in T Cells from Patients with Systemic Lupus Erythematosus J. Immunol., October 15, 2002; 169(8): 4147 - 4152. [Abstract] [Full Text] [PDF]
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 P. Sassone-Corsi Editorial: Never Enough--On the Multiplicity and Uniqueness of Transcriptional Regulators in Postmeiotic Male Germ Cells Endocrinology, May 1, 2002; 143(5): 1575 - 1577. [Full Text] [PDF]
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G. Stelzer and J. Don Atce1: A Novel Mouse Cyclic Adenosine 3',5'-Monophosphate-Responsive Element-Binding Protein-Like Gene Exclusively Expressed in Postmeiotic Spermatids Endocrinology, May 1, 2002; 143(5): 1578 - 1588. [Abstract] [Full Text] [PDF]
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 S. A. Shell, C. Fix, D. Olejniczak, N. Gram-Humphrey, and W. H. Walker Regulation of Cyclic Adenosine 3',5'-Monophosphate Response Element Binding Protein (CREB) Expression by Sp1 in the Mammalian Testis Biol Reprod, March 1, 2002; 66(3): 659 - 666. [Abstract] [Full Text] [PDF]
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B. M. Sanborn Editorial: Increasing the Options--New 3',5' Cyclic Adenosine Monophosphate (cAMP)-Responsive Promoters and New Exons in the cAMP Response Element Modulator Gene Endocrinology, November 1, 2000; 141(11): 3921 - 3922. [Full Text] [PDF]
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