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Institut de Génétique et de Biologie Moléculaire et Cellulaire 67404 Illkirch, Strasbourg, France
Address all correspondence and requests for reprints to: Paolo Sassone-Corsi, Institut de Génétique et de Biologie Moléculaire et Cellulaire, B.P. 163, 67404 Illkirch, Strasbourg, France. E-mail: . paolosc{at}titus.u-strasbg.fr
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Introduction |
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 Top Introduction References |
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Most of our current knowledge on transcriptional regulation comes from studies performed in somatic cells (5). Although many examples of tissue- and cell-specific transcriptional mechanisms have been described, the events in testis carry a number of additional wrinkles. The presence of germ cell-specific factors is evidenced by the large number of promoters that have been found to have a pattern of activity restricted to testis (1, 2). A tentalizing feature is that a significant number of the genes activated postmeiotically contain a CRE (cAMP-responsive element) in their regulatory promoter region (6). This is of interest considering the importance that cAMP-dependent signaling has in spermatogenesis. Indeed, some specific isoforms of both the adenylyl cyclase and cAMP-dependent kinase have been found in testis (7, 8, 9).
The CRE consensus site is constituted by the canonical 8-bp palindromic sequence TGACGTCA. Factors of the CREB (cAMP-responsive element binding protein) family were originally identified as binding CREs and to functions as activators that respond directly to the cAMP-dependent signaling pathway via phosphorylation by the PKA (10). This family comprises a large number of proteins encoded by the CREB, CREM (cAMP-responsive element modulator) and ATF-1 (activating transcription factor 1) genes. In reality, these factors are final effectors of a number of signaling pathways, stimulated not only by cAMP, but also calcium, growth factors, and stress signals (10). Phosphorylation of CREB proteins allows recruitment of CBP (CREB-binding protein), a large coactivator with histone acetyl-transferase activity that contacts the general transcriptional machinery (10).
CREB, CREM, and ATF-1 belong to the basic domain-leucine zipper (bZip) transcription factor class of proteins and act as dimers (10). They are able to heterodimerize with each other but only in certain combinations. Thus, a dimerization code exists that seems to be a property of the leucine zipper structure of each factor. Whereas the zippers of the oncoproteins Jun and Fos contain five leucines, all proteins of the CREB family contain four leucines. The CREB-like factors are able to form all possible heterodimers, drastically increasing the combinatorial association potential, and thereby eliciting a number of likely different regulatory functions.
An important feature of factors of the CREB family is their almost ubiquitous expression in all tissues (10). There is, however, a notable exception: CREM expression in the male germ cells (11). At early meiotic stages of prophase I in the mouse, antagonists forms of both CREB (
and 
, lacking the bZIP domain; 12) and CREM (, ß, and , lacking the activation domain; 11) are present. While the physiological function of the CREB isoforms is yet undetermined, CREM is the subject of a remarkable developmental switch in expression: it is highly abundant in adult testis, while in prepubertal animals it is expressed at very low levels (11, 13). It is by processes of alternative splicing and alternative polyadenylation that the various CREM isoforms are expressed at different times during the differentiation program of the germ cells (11, 13, 14). While in prepubertal testis only the repressor forms (, ß, and ) are detected at low levels, the abundant CREM transcript in the adult encodes exclusively the activator form CREM
. Thus, the CREM developmental switch constitutes a reversal of function (11, 13, 15).
The CREM activator is essential for spermatogenesis. Targeted ablation of the CREM gene results in a complete block of the differentiation program at the first step of spermiogenesis (16, 17). Early differentiation and stem cell renewal occur normally, in accordance with previous observations showing that the CREM protein accumulates only at the round spermatid and later stages (6). The stringent requirement for CREM is manifested by the lack of maturation of the germ cells and by their entry into the cell death pathway. Indeed, deletion of CREM causes a 10-fold increase in the number of apoptotic germ cells, indicating that the death-interfering function of CREM is restricted to cells undergoing the second division of meiosis (16). This specialized function in testis physiology is unique to CREM, as genetic disruption of CREB results in embryonic lethality, whereas mutation of ATF-1 has no apparent effect (18).
How CREM can exert its function in determining critical steps of spermiogenesis? A number of postmeiotically expressed genes (e.g. transition protein-1, calspermin) contain CREM-binding sites in their promoters and were found to be direct targets of this transcriptional activator (15). In this scenario, CREM would need to interact with additional components of the transcriptional machinery which probably have specialized functions for germ cells. Indeed, association has been found with TFIIA (De Cesare, D., G. M. Fimia, and P. Sassone-Corsi, unpublished), which itself is known to interact with a TBP-like factor, TLF, whose essential function in spermiogenesis was also demonstrated by genetic mutation in the mouse (19, 20, 21).
As an unexpected actor entering a stageplay, a new element has appeared in this picture. Stelzer and Don report in this issue (22) the cloning of a new CREB-like gene whose expression is developmentally regulated during mouse spermatogenesis. The gene, named Atce1, was found by screening a testis library with a two-hybrid approach where the Tctex2 coding sequence was used as bait. Interestingly, Tctex2 is a gene that was identified as a meiotic element evolutionary conserved between yeast and mouse. Atce1 expression in germ cells appears restricted to the mid/late round spermatids—so the same cells where CREM was shown to exert its action.
Several features of Atce1 are of interest. Differently from CREB, CREM, and ATF-1—which all have the bZip domain in the C-terminus—the putative Atce1 aminoacidic sequence would encode a protein with the bZIP domain placed in the middle of the protein (22). This relatively uncommon feature is shared by LZIP and CREB-H, two proteins not expressed in testis and whose function has not been fully explored. Another protein with the bZip domain in a similar position is Fos, which participate in transcription factor AP-1 by associating with Jun—that instead has the bZip in the C-terminus. Importantly, as Fos is not able to homodimerize, one wonders how Atce1 may function, as monomer, homodimer, or whether a partner is involved at some time. The experiments presented by Stelzer and Don (22) are not conclusive on this point, but they suggest that Atce1 has anyway a functionally distinct action. First, although expressed in the same cells than CREM (mid/late spermatids), Atce1 is not able to complement its function in the CREM-null mice. Second, based on DNA-binding experiments, it appears that Atce1 does not bind CRE sequences. Thus, as CREM is a potential partner of Atce1, it would seem that in a putative heterodimer Atce1 would have the capacity of modifying the DNA-binding properties of CREM. Atce1 is found to bind to a NF-
B regulatory site, an interesting finding considering that NF-B activity has been described as being modulated from spermatocytes to spermatids (23, 24). It will be of interest to identify by DNA binding site-selection strategies whether Atce1, alone or in combination with putative partners, recognizes other promoter elements.
The lack of anti-Atce1 antibodies does not allow one to determine in which germ cells the protein is expressed and its intracellular localization. While this information will be certainly useful for further understanding of Atce1 function, it is interesting that the protein contains a nuclear localization domain (NLS) located within the bZip region. Most proteins of the bZip class, and certainly all members of the CREB/CREM family, have a NLS and have been found to be consistently nuclear. Thus, Atce1 contains the features of a nuclear transcriptional effector and the elucidation of its physiological function would be of undoubtful interest. In this respect, it is important to note that the putative coding sequence of Atce1 contains two glutamine-rich (Q1 and Q2) regions that are likely to correspond to the activations domains as described in CREB and CREM (10). Notably, the Q-rich domains in Atce1 are on both sides of the bZip region, in a position unique for this class of transcription factors. In contrast, the Q1 and Q2 domains in CREB and CREM are located in the N-terminal half of the molecule, flanking the phosphorylation box, the site of modification by various kinases (10). These notions suggest that CREM and Acte1 may function in a similar manner with respect to interactions with putative coactivators.
Surprisingly, CREM does not seem to be phosphorylated in germ cells; finding that raised the question of how activation may be working. The answer to this puzzle was found by the identification of a testis-specific activator of CREM that is able to overcome the phosphorylation requirement (25). This tissue-specific coactivator, ACT (activator of CREM in testis) interacts with CREM in spermatids. ACT is a factor belonging to the class of LIM (from the proteins Lin-11, Isl-1, and Mec-3)-only proteins with a characteristic organization of four and half LIM domains. These structural motifs are composed of two adjacent zinc fingers and are known to be involved in protein-protein interactions. ACT expression is testis-specific and temporally coordinated with CREM during germ cell differentiation. Upon binding to the CREM activation domain, ACT powerfully stimulates CREM transcriptional activity in a phosphorylation- and CREB binding protein-independent manner (25). Could Acte1 also interact with ACT, or with other members of this family of transcriptional coactivators? Another ACT-like protein, FHL-4 (four and half LIM-4), for example, is also highly expressed in testis but it does not interact with CREM (26). Further experimentation is needed to provide answers to these questions. In particular, it will be important to establish the regulatory function of Acte1—a likely activator of transcription—and to identify its target genes. This will lead to determine its functional relationship with the other CREB-like proteins in testis and to establish its biological function in the developmental process of spermatogenesis. Future analyses are likely to provide some fascinating new insights.
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Footnotes |
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Received March 12, 2002.
Accepted for publication March 13, 2002.
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References |
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TopIntroductionReferences |
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isoforms expressed by two newly identified cAMP-responsive promoters active in the testis. Endocrinology 141:3923–3930B in mammalian testis. Mol Endocrinol 12:1696–1707B is developmentally regulated during spermatogenesis in mice. Dev Dyn 219:333–340[CrossRef][Medline]
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