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Molecular Endocrinology, doi:10.1210/me.2003-0280
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Molecular Endocrinology 17 (12): 2554-2565
Copyright © 2003 by The Endocrine Society

Transcriptional Control in Male Germ Cells: General Factor TFIIA Participates in CREM-Dependent Gene Activation

Dario De Cesare, Gian Maria Fimia, Stefano Brancorsini, Martti Parvinen and Paolo Sassone-Corsi

Institut de Génétique et de Biologie Moléculaire et Cellulaire (D.D.C., G.M.F., S.B., P.S.-C.), 67404 Illkirch, Strasbourg, France; and Department of Anatomy (M.P.), University of Turku, 20520 Turku, Finland

Address all correspondence and requests for reprints to: Paolo Sassone-Corsi, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries, 67404 Illkirch, Strasbourg, France. E-mail: paolosc{at}igbmc.u-strasbg.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of gene expression in haploid male germ cells follows a number of specific rules that differ from somatic cells. In this physiological context, transcriptional control mediated by the activator CREM (cAMP-responsive element modulator) represents an established paradigm. In somatic cells activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CBP (cAMP response element binding protein-binding protein). In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, ACT (activator of CREM in testis), which confers a powerful, phosphorylation-independent activation capacity. In addition to specialized transcription factors and coactivators, a variety of general factors of the basal transcriptional machinery, and their distinct tissue-specific isoforms, are highly expressed in testis, supporting the general notion that testis-specific gene expression requires specialized mechanisms. Here, we describe that CREM interacts with transcription factor IIA (TFIIA), a general transcription factor that stimulates RNA polymerase II-directed transcription. This association was identified by a two-hybrid screen, using a testis-derived cDNA library, and confirmed by coimmunoprecipitation. The interaction is restricted to the activator isoforms of CREM and does not require Ser117. Importantly, CREM does not interact with TFIIA{tau}-ALF, a testis-specific TFIIA homolog. CREM and TFIIA are expressed in a spatially and temporally coordinated fashion during the differentiation program of germ cells. The two proteins also colocalize intracellularly in spermatocyte and spermatid cells. These findings contribute to the understanding of the highly specialized rules of transcriptional regulation in haploid germ cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A NUMBER OF transcription factors integrate physiological responses by responding to activation of intracellular transduction pathways. By doing so, they generate programs of gene expression that are frequently unique to a specific cell type and that may determine dynamic decisions on whether to proliferate, differentiate, or die. It appears that the rules governing gene transcription and chromatin remodeling in haploid germ cells are indeed unique as they drastically differ from somatic cells (1). Studies on the activator CREM [cAMP-responsive element (CRE) modulator] have contributed to the identification of some essential features of male germ cell-specific transcriptional complexes (2).

In somatic cells, transcription factors of the cAMP response element binding protein (CREB) family regulate gene expression in response to a variety of signaling pathways (2). These factors bind to DNA sequences known as CREs through their C-terminal bZip domain (3, 4). The N-terminal half of CREB and CREM contains a modular activation domain (AD) composed of two independent regions (5, 6). One region comprises two glutamine-rich domains, Q1 and Q2, flanking a second region, the phosphorylation box (P-box), also known as the kinase-inducible domain (KID). The P-box contains a key serine residue (Ser133 in CREB, Ser117 in CREM) that is phosphorylated by kinases stimulated by cAMP, calcium, growth factors, and stress signals (2, 7, 8). Phosphorylation at this serine is required for interaction with the coactivator CREB binding protein (CBP) and consequent transcriptional activation of CRE-containing promoters (9).

The products of the CREM gene are regulated in a tissue-specific manner and play an essential role during the spermatogenesis differentiation process (10). Spermatogenesis corresponds to a hormonally regulated sequence of highly coordinated developmental events that lead to the formation of haploid spermatozoa from precursor diploid stem cells. The CREM gene is highly and specifically expressed in post-meiotic male cells where it regulates a number of genes involved in the process of spermatogenesis (10, 11, 12). CREM has an essential role in spermatogenesis as demonstrated by its targeted mutation in the mouse (13, 14). Importantly, in contrast to somatic cells, CREM does not appear to be phosphorylated in male germ cells but associates with ACT (activator of CREM in testis), a member of the LIM-only class of proteins that has an intrinsic transcriptional activity (15). Proteins of the LIM class contain a domain first identified in the proteins encoded by the Lin-11, Isl-1, and Mec-3 genes. Thus, CREM-mediated control of post-meiotic genes is provided by the interaction with ACT, bypassing the requirements for interaction with CBP. This finding points to a tissue-specific modulation mechanism of CREM transcriptional activity. Interestingly, other LIM-only proteins, such as FHL-2 and FHL-3, have been shown to function as phosphorylation-independent coactivators of CREM and CREB (16).

Several lines of evidence indicate that general transcription factors may also be differentially regulated in germ cells. For example, TBP (TATA-binding protein) accumulates in early haploid germ cells at higher levels than in any other somatic cell type (17, 18). More importantly, the TBP-related factor TLF (TBP-like factor) was shown to be highly expressed in testis and to exert a specialized function in male germ cells. Indeed, targeted disruption of the TLF gene leads to a block in spermiogenesis that is reminiscent of the CREM-null mice (19). It has been proposed that TLF substitutes TBP in initiation complexes associated to TATA-less promoters (20), and it is intriguing that a number of post-meiotic CREM-target genes indeed lack a TATA element (21).

In addition to TLF, also transcription factor (TF) IIB, TFIIE{alpha}, and RNA polymerase II subunits were found to be overexpressed in testis (22, 23), whereas both TFIIA{alpha}/ß and TFIIA{gamma} have been described to be abundant in human testis (24). These remarkable features are consistent with the general notion that male germ cells display a high overall transcription level. In addition, some homologs of general transcription factors are testis specific. For example TAF7L, a paralog of TFIID subunit TAF7, associates with TBP in pachytene and haploid cells, and is developmentally regulated (25). Another interesting case is represented by ALF (TFIIA{alpha}/ß-like factor), a testis-specific homolog of TFIIA, whose expression is 50-fold higher in human testis than in any other tissue (26, 27). Thus, several observations indicate that, in addition to specialized transcription factors and coactivators, factors of the basal transcriptional machinery may play unique roles in testis gene expression. To date, however, little information is available on the molecular and functional interaction that may exist between transcriptional activators and general factors in haploid germ cells.

In this report, we show that CREM specifically interacts with the general transcription factor TFIIA. This interaction requires the AD of CREM and appears to be independent of Ser117 phosphorylation. We also show that TFIIA expression is regulated during testis development after an expression profile highly similar to CREM and that the two proteins are expressed in the same germ cellular types. These findings provide new insights into the molecular mechanisms by which CREM exerts its function in the regulation of post-meiotic gene transcription.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CREM Interacts with TFIIA in Testis
The activator CREM is highly expressed in male germ cells where it controls the transcription of various post-meiotic genes (28). In an attempt to investigate the molecular mechanisms of CREM function in testis and identify putative testis-specific partners, we performed a two-hybrid screen of a murine testis-derived cDNA library using the CREM AD as a bait (Fig. 1AGo). Using the same approach, we had previously identified ACT, a LIM-only protein exclusively expressed in spermatids, that functions as a tissue-specific coactivator of CREM (15). Here we describe another clone, different from ACT, which specifically and efficiently interacts with CREM AD, both in nutritional selection (not shown) and ß-galactosidase reporter assays (Fig. 1BGo). Data bank searches revealed that this clone, denominated 9H, corresponds to part of the sequence encoding for the TFIIA{alpha}ß-subunit of the general TFIIA [nucleotides (nt) 1–822, amino acids (aa) 1–274; see Fig. 1BGo, lower panel]. TFIIA has been shown to stimulate RNA polymerase II-specific transcription and is encoded by two genes, TFIIA{alpha}ß and TFIIA{gamma}, highly conserved between human and yeast (29). The product of the TFIIA{alpha}ß gene is an {alpha}ß pro-polypeptide (see lower panel in Fig. 1BGo) cleaved proteolitically to generate the two largest subunits of TFIIA. These associate with the product of the TFIIA{gamma} gene to form a transcriptionally active heterotrimeric complex (29). Sequence alignment revealed that clone 9H contained part of the TFIIA{alpha}ß open reading frame, including the whole region encoding the TFIIA{alpha}-subunit and most of the nonconserved region between the {alpha} and ß domains (white box in the scheme in Fig. 1BGo). Thus, here we identify clone 9H to TFIIA{alpha}.



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  		Fig. 1. Isolation of TFIIA in Testis by Yeast Two-Hybrid Assay

A, Gal4-DBD/CREM fusion used as a bait and the CREM AD structure. Q1 and Q2, Glutamine-rich domains. P-box, Phosphorylation box. B, The CREM AD specifically interacts with TFIIA{alpha}/ß. Top, ß-Galactosidase levels in yeast transformed with the indicated constructs. GBT, Yeast expression vector for the Gal4 DNA binding domain; GAD, yeast expression vector for the Gal4 activation domain; LAM5, yeast expression vector for Gal4-DBD-laminin 5 fusion product. Bottom, Schematic representation of TFIIA{alpha}ß, ALF, and 9H clone; length of the proteins is indicated in terms of aminoacid numbers; {alpha} and ß domains are indicated, white boxes indicate the nonconserved regions. C, Selectivity of CREM/TFIIA interaction: the CREM AD does not associate with the testis counterpart of TFIIA, ALF. Yeast cells were cotransformed with the indicated plasmids. Results from the ß-galactosidase assay are reported in Miller units as a mean of at least two independent experiments each done in triplicate. As a negative control, the Gal4-AD encoding vector was cointroduced in yeast with the Gal4-DBD-CREM fusion. TFIIA{gamma} and ALF ORFs were fused to the Gal4 AD and equal level of expression were checked by Western analysis of yeast extract by using an anti-Gal4 AD antibody. D, TFIIA{alpha} strongly interacts with glutamine-rich activation domains. Yeast cells were cotransformed with the indicated plasmids (CREM, CREB, c-Jun, and Sp1 were fused to the Gal4 DBD) and results from the ß-galactosidase assay reported as fold activation. Fold activation was calculated as a ratio between the values obtained in the presence of the Gal4-TFIIA{alpha} and various Gal4-DBD fusion proteins, respectively, and the value obtained in the presence of the Gal4-DBD fusions alone.

 
Highly Specific Interaction with the TFIIA{alpha}-Subunit
To gain further insight into the physiological significance of the CREM-TFIIA interaction, we studied its specificity by analyzing various TFIIA isoforms. Because TFIIA has been reported to be active as a trimeric complex, we tested whether the {gamma}-subunit was also able to interact with the CREM AD. We fused the TFIIA{gamma}-subunit open reading frame (ORF) to the Gal4-AD and expressed the fusion product in yeast, in combination with the Gal4DBD-CREM AD fusion protein (Fig. 1AGo). Unlike the TFIIA{alpha}-subunit, the {gamma}-subunit was unable to drive the transcription of the ß-galactosidase reporter (Fig. 1CGo), indicating that the two proteins do not associate directly.

A TFIIA testis-specific homolog, named ALF (TFIIA{alpha}/ß-like factor), has been described (27). ALF is a functional counterpart of TFIIA{alpha}/ß that, together with TFIIA{gamma}, can stabilize TBP/TATA element interaction and thereby support RNA polymerase II-dependent transcription in vitro. As ALF is expressed in testis at levels higher than in other tissues, it was important to establish whether it could interact with CREM. We fused the Gal4AD to the ALF open reading frame (see Fig. 1BGo, lower panel) and tested the ability of this chimeric protein to associate with CREM AD. The results from the two-hybrid assay showed no significant interaction between ALF and CREM AD (Fig. 1CGo), compared with the robust induction of ß-galactosidase activity observed in the presence of TFIIA{alpha}. Thus, TFIIA{alpha} and its testis-specific homolog ALF do not share the property of associating with CREM.

To further confirm and extend the specificity of the CREM-TFIIA interaction, we studied the capacity of ADs derived from different transcriptional activators. We fused the ADs to the Gal4-DBD and tested interaction with the Gal4AD-TFIIA{alpha} fusion protein (Fig. 1DGo). CREB AD was chosen because of its high homology to CREM, whereas Sp1 bears a glutamine-rich domain showing no significant homology to glutamine-rich domains of CREM and CREB (30) (and our unpublished observations); finally, c-Jun does not contain apparent glutamine-rich motif within its AD (Ref. 31 and our unpublished observations). While CREB and Sp1 ADs were able to associate with TFIIA as efficiently as CREM AD, the Gal4DBD-c-Jun AD fusion failed to activate the reporter gene transcription in the presence of TFIIA{alpha} fusion product (Fig. 1DGo). Thus, the interaction of TFIIA{alpha} with CREM is highly specific. Furthermore, TFIIA{alpha} seems to interact with factors containing glutamine-rich motifs, although their presence does not seem to be uniquely essential for interaction.

To define the CREM domains involved in the interaction with TFIIA, we tested different Gal4DBD-CREM AD deletions (Fig. 2AGo, left panel) for their efficacy in associating with the Gal4AD-TFIIA{alpha} chimera. The Q1 and Q2 glutamine-rich domains were both needed for efficient interaction with TFIIA (Fig. 2AGo, right panel) because the CREMß isoform was inert in two-hybrid assay as compared with CREM{tau} protein. However, the lack of Q2 appeared to be more detrimental (compare CREM{tau}1 and CREM{tau}2). The P-box region also appears to contribute to the strength of association because the isoforms retaining Q1 and Q2 modules, but lacking the P-box domain (CREMQ1 and CREMQ2 in Fig. 2AGo), were completely inactive compared with the CREM{tau}2 construct, which showed a residual activity. Taken together, these results indicate that multiple determinants within the CREM AD are needed for full functional interaction with TFIIA{alpha}.



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  		Fig. 2. CREM-TFIIA Interaction Is Specific to CREM Activator Isoforms

A, CREM isoforms (schematically represented in left panel: P-box, phosphorylation box; Q1 and Q2 glutamine rich-domains are indicated) were fused to Gal4-DBD and coexpressed in yeast cells together with the Gal4-AD-TFIIA{alpha} fusion protein. Results are reported in Miller Units. B, CREM/TFIIA interaction is independent of phosphorylation of CREM at Ser117. Right, Sequence of the peptide, within the CREM P-box domain, encompassing Ser117 compared with its mutagenized counterpart (CREMAla117). Left, Results from ß-galactosidase assay (Miller Units) upon when coexpression, in yeast, of the Gal4-AD-TFIIA{alpha} fusion with the wild-type Gal4-DBD-CREM fusion protein and its Ala117 mutated version, respectively. Fold activation is calculated as a ratio between the values obtained when Gal4-DBD-CREM and Gal4-AD-TFIIA fusions were expressed together with respect to the value obtained in the presence of the Gal4-DBD-CREM and Gal4-DBD-CREMAla117 products alone.

 
Association Is Independent of Ser117
Phosphorylation of Ser117 within the CREM AD is essential for association with CBP (9, 32), whereas association with ACT has been shown to confer transcriptional activity on CREM in a phosphorylation and CBP-independent manner (15). On the basis of these notions and because the interaction of CREM with TFIIA seems to require the presence of the P-box (Fig. 2AGo), we wondered whether the CREM-TFIIA{alpha} association would be dependent on Ser117. To test this possibility, Ser117 was converted into an alanine in the context of the Gal4DBD-CREM AD construct (Fig. 2BGo, right panel); this mutated construct was used in a two-hybrid assay in combination with the Gal4AD-TFIIA{alpha}. The Ser>Ala mutation has no effect on the transcription of a ß-galactosidase reporter gene (Fig. 2BGo, left panel), thus indicating that, similarly to the CREM-ACT association (15), the CREM-TFIIA interaction is independent of the presence of Ser117 within the P-box.

TFIIA Protein Domains Involved in CREM Interaction
Next, we wished to identify the molecular determinants of TFIIA{alpha} responsible for interaction with CREM. For this, we generated various TFIIA{alpha} deletion mutants (Fig. 3Go, left panel). All mutations impaired the interaction with CREM, albeit to different extents. Indeed, internal deletions introduced both in the highly conserved {alpha} domain (Fig. 3Go, black box in left panel) and the nonconserved region (Fig. 3Go, white box in left panel) drastically impaired CREM association. A 127-aa truncation in the TFIIA carboxy terminus severely affected, although did not abolish, interaction with CREM. These results indicate that the CREM-TFIIA interaction is stabilized by the presence of different regions within the TFIIA{alpha}-subunit.



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  		Fig. 3. TFIIA Deletions Interfere with CREM Interaction

Left, Schematic drawing of TFIIA deletion mutants. Amino acid numbers corresponding to portions of the TFIIA protein are indicated. TFIIA mutants were fused to the Gal4-AD. The {alpha} and nonconserved domains of TFIIA are also indicated (black and white boxes, respectively). Right, Results (Miller units) from ß-galactosidase assay using the Gal4-DBD-CREM plasmid in combination with the various Gal4-TFIIA deletions.

 
CREM and TFIIA Associate in Mammalian Cells
The interaction between CREM and TFIIA{alpha} was further investigated by coimmunoprecipitation experiments after coexpression of the two proteins in mammalian cells. COS cells were cotransfected with a Myc-tagged TFIIA{alpha} expression vector in combination with a CREM expression vector. Protein extracts were immunoprecipitated by using an anti-CREM antibody. Immunoprecipitated products were resolved by SDS-PAGE and revealed by Western blot analysis using anti-Myc antibodies. The tagged TFIIA{alpha} protein was coimmunoprecipitated by CREM antibodies only when expressed concomitantly with CREM, whereas no Myc-TFIIA{alpha} was detected in the immunoprecipitated complexes when either the Myc-TFIIA{alpha} or the CREM expression vector were transfected alone in COS cells (Fig. 4Go).



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  		Fig. 4. TFIIA Associates with CREM in Vivo

COS cells were cotransfected with pSVCREM and pCS2Myc-tagged TFIIA expression vectors. Cells were harvested 48 h after transfection, and lysates were subjected to immunoprecipitation with CREM antibodies. Whole extracts (left) and immunocomplexes (right) were analyzed by Western blotting using both anti-CREM and anti-Myc antibodies as indicated.

 
TFIIA and CREM Display Overlapping Expression Profiles
We wished to examine the expression of TFIIA{alpha} in different tissues as compared with its levels in testis. To this aim, we raised a rabbit polyclonal antibody ({alpha}293) against a peptide corresponding to aa 255–273 of the TFIIA{alpha} protein. The TFIIA protein was expressed at detectable levels in lung, spleen, and in ovary, whereas it appeared to be more abundant in testis by at least 4-fold (Fig. 5AGo).



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  		Fig. 5. Tissue-Specific and Developmentally Regulated Expression of TFIIA

A, Expression of TFIIA protein in different adult mouse tissues. Extracts from the indicated tissues were prepared in boiling Laemmli buffer and normalized by Coomassie blue staining to ensure equal loading of total proteins. Western blot was probed with anti-TFIIA antibodies. B, Expression of TFIIA during testis development. Ten micrograms of total RNA extracted from testes of mice at different ages post birth (as indicated) were analyzed by ribonuclease protection assay, using TFIIA, CREM, and ACT specific riboprobes. A ß-actin riboprobe was used as an internal control. C, Ten micrograms of total RNA extracted from testes of adult wild-type and CREM null mice (CREM -/-) were analyzed using a TFIIA-specific riboprobe. D, Immunodetection of TFIIA, CREM and ACT in specific testis cell types. Protein extracts from the indicated germ and somatic cell lines from mouse testis were prepared in boiling Laemmli buffer and loaded onto a 10% SDS-PAGE. Western blots were probed with the indicated antibodies (left of each panel). Red Ponceau staining is shown to represent equal loading of proteins.

 
Two approaches were used to investigate TFIIA expression in the testis in more detail. First, we analyzed the time of appearance of TFIIA transcript during postnatal testis development. Indeed, during the first wave of spermatogenesis, germ cells are synchronized in their development and the timing of appearance of each cell type is specifically defined. Ribonuclease protection analysis of RNA prepared from 1- to 4-wk-old mouse testes revealed that TFIIA expression is low at the first week after birth, whereas a robust increase occurs at the third week, at the time of accumulation of late pachytene spermatocytes and round spermatids. Expression remained elevated at 4 wk and in the adult, thus showing a profile highly similar to CREM (Fig. 5BGo), although the increase in TFIIA transcript levels seems to precede that of CREM. Maximum expression of ACT, the testis-specific CREM coactivator (15), also occurred from the third to the fourth week. In conclusion, TFIIA expression appears to be at least in part temporally coordinated with that of CREM and ACT, being present in both late meiotic and post-meiotic cells. That TFIIA expression precedes CREM was confirmed by using testes from CREM -/- mice, where TFIIA expression is still observed (Fig. 5CGo).

The distribution of TFIIA was further analyzed using whole protein extracts prepared from purified germ and Sertoli mouse cells. TFIIA is present in both spermatocytes and spermatids, whereas it is only weekly expressed in somatic Sertoli cells (Fig. 5DGo). CREM is coexpressed in the same cellular types as TFIIA, whereas ACT is predominant in spermatids. Thus, TFIIA is predominantly expressed in germinal cells and its expression profile partially overlaps those of CREM and ACT.

The developmentally regulated expression of TFIIA was further investigated by isolating segments of rat seminiferous tubules corresponding to each developmental stage by transillumination-assisted microdissection (33). Segments were subjected to both Western analysis and immunostaining with antibodies for TFIIA and CREM. TFIIA expression appeared to be regulated during the spermatogenic wave with increased levels from stages VI to IX–XI (Fig. 6AGo). The developmental distribution of CREM activator isoforms (CREM{tau}, CREM{tau}1, and CREM{tau}2) is also regulated, showing a sharp peak of expression overlapping with the increase observed for TFIIA.



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  		Fig. 6. Localization of CREM and TFIIA in Germ Cells

A, Rat stage-specific protein extracts (indicated above each lane) were prepared and used for Western analysis. Western blots were probed with the indicated antibodies (left of each panel). Red Ponceau staining is shown to represent equal loading of proteins. B, Expression of CREM and TFIIA in specific germ cells. Rat and mouse squash preparations (as indicated) from fixed sectioned seminiferous tubules were probed with both anti-CREM and TFIIA antibodies (left panels of each series). Immunodetection was performed through confocal microscopy. PS, Pachytene spermatocyte; RS, round spermatid. The middle and right panels show the corresponding DAPI (4',6-diamidino-2-phenylindole)-stained DNA and merged images, respectively. Magnification, x400.

 
Intracellular Colocalization of TFIIA and CREM
Detailed confocal microscopy studies were performed on squash preparations prepared from both rat and mouse seminiferous tubules to address the question of the intracellular localization of TFIIA{alpha} and CREM. Analysis by immunostaining of pachytene spermatocytes (PS) and round spermatids (RS) at various stages of the spermatogenic wave reveals that, both in the rat and mouse, TFIIA localizes in the nuclei of pachytene spermatocyte and round spermatid cells, as shown in Figs. 6BGo and 7AGo, whereas no signal was observed in spermatogonia and mature spermatozoa (see Fig. 7AGo). In pachytene spermatocytes TFIIA nuclear distribution appears to be concentrated in speckles which are excluded from heterochromatic bodies (Fig. 6BGo). This pattern is conserved in round spermatids where TFIIA surrounds the heterochromatic chromocenter structure. Similarly to TFIIA, CREM nuclear distribution in pachytene spermatocytes is also in speckles (6B). In round spermatids, CREM became more abundant and its distribution became more diffuse, although still excluded from heterochromatin. Elevated TFIIA expression in spermatocytes was seen mostly at stages VII–XII, whereas at stages V-VIII TFIIA was also expressed in spermatids (see Fig. 7AGo). CREM was preferentially localized in spermatids at stages V–VIII, whereas accumulation in spermatocytes became prominent from stage XI (Fig. 7AGo). Accumulation of both protein in spermatids seems to occur at stages V–VIII, although at these stages TFIIA was also expressed in spermatocytes where little CREM expression was detected. Thus, both TFIIA and CREM follow a similar dynamic distribution during the differentiation of male germ cells, being always associated with the transcriptionally active euchromatin. The developmentally regulated distribution of the two proteins along the spermatogenic wave, based on our confocal microscopy studies and immunohistochemistry analysis, is schematically presented in Fig. 7BGo.



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  		Fig. 7. Distribution of CREM and TFIIA during the Spermatogenic Wave

A, Immunodetection of CREM and TFIIA in developing male germ cells. CREM and TFIIA are detected in microdissected segments of tubules from various stages as indicated on the left; left panels of each column show immunodetection with anti-CREM and anti-TFIIA antibodies, whereas middle and right panels show DAPI (4',6-diamidino-2-phenylindole)-stained DNA and merged images, respectively. Magnification, x110. B, Summary of TFIIA and CREM expression profile during spermatogenesis. The spermatogenic wave is schematized and the representative cell types are indicated. Spermiogenesis steps are indicated by numbers 1–16. Expression of TFIIA and CREM is shown in their respective panels by the red coloring. SG, Spermatogonia; PL, preleptotene; L, leptotene; Z, zygotene; P, pachytene; D, diakinetic; ES, elongating spermatids; MS, mature spermatozoa.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The identification of a number of variants of general transcription factors and coactivators that play specific roles during differentiation and embryonic development has provided important leads toward the understanding of tissue-specific gene expression. An important level of control operates at the preinitiation complex assembly. An example is represented by the group of TBP-like factors, TRF1/2 and TLF, identified in Drosophila and in mouse, respectively. TRF (TBP-related factor) has been characterized as a tissue-specific form of TBP (34, 35), whereas TLF (TBP-like factor) is highly expressed in testis where it plays a crucial role during spermatogenesis (19, 36). An additional example is the TFIIA{tau} (or ALF) factor, a testis-derived variant of the general transcription factor TFIIA. This variant has been described as a functional homolog of the human general transcription factor TFIIA{alpha}/ß that may be uniquely important to testis-specific transcription regulation (26, 27). Finally, a number of general transcription factors display unique patterns of expression in testis (37).

We have reported the interaction between the testis-specific activator CREM and the general transcription factor TFIIA. The assembly of the preinitiation complex and transcription initiation by RNA polymerase II are known to involve TFIIA. It seems that in addition to stabilizing TBP interaction with the TATA element and TFIID contacts with promoter and initiator sequences, TFIIA also serves as a cofactor for several activators and coactivators (27, 37). In this respect, our observations support the notion that TFIIA may also function as a versatile link between the preinitiation complex and upstream activators.

The interaction between CREM and TFIIA occurs preferentially with the CREM{tau} activator isoform, suggesting that the other known activator isoforms (CREM{tau}1 and CREM{tau}2) may contact the basal transcription machinery in different ways. The CREMß isoform, as well as ICER (not shown), both repressors of CRE-dependent transcription, do not associate with TFIIA. CREMß lacks both Q1 and Q2 glutamine-rich domains, which have been shown to be essential for full CREM transcriptional activity. The importance of glutamine-rich domains in the interaction is confirmed by the observation that the Sp1 activation domain, enriched in glutamine residues, also strongly interacts with TFIIA (Fig. 1DGo). However, the CREM-TFIIA association does not seem merely due to a generic affinity of TFIIA for glutamine-rich motifs. First, the lack of one of the two Q modules in the CREM AD is detrimental to the interaction (Fig. 2AGo). In addition, the presence of the P-box is important for the efficiency of CREM-TFIIA interaction, although the presence of the phosphoacceptor site Ser117 within the KID domain does not appear to be necessary (Fig. 2BGo). Taken together, these results may reflect a complex mode of interaction for which the full conformation of CREM AD or multiple determinants within the CREM AD are required to stabilize the association between the two proteins.

Although the presence of the P-box within the CREM AD is essential for efficient interaction with TFIIA, it is apparent that the mechanism by which the two proteins associate is independent of Ser117 (Fig. 2BGo). A phosphorylation-independent mechanism has been described for the coactivator function of ACT, a testis-specific partner of CREM (15). Thus, the interaction of TFIIA with the unphosphorylated form of CREM is in keeping with the evidence that an alternative pathway of transcriptional activation by CREM exists in testis.

TFIIA is encoded by two highly conserved genes ({alpha}ß and {gamma}) and can be isolated from metazoan cells as a heterotrimeric complex (29). This is formed by two largest subunits, {alpha} and ß, which are produced by a specific proteolytic cleavage of the {alpha}ß polypeptide (38, 39). The small subunit, {gamma}, is encoded by a different gene (40). In the budding yeast Saccharomyces cerevisiae, TFIIA functions as a heterodimer, the large subunit (TOA1) not being proteolitically cleaved (41, 42). The human TFIIA{alpha}ß and the yeast TOA1 genes share significant sequence similarity at the amino- ({alpha}) and carboxyl-terminal (ß) domains, whereas the spacer region between these two domains shows very little homology (Fig. 1BGo). Our data demonstrate that the {alpha}-subunit of TFIIA is sufficient for interaction with CREM AD (Fig. 1Go, B and C), the ß-subunit being apparently dispensable, although it cannot be excluded that it may play a significant role in stabilizing CREM-TFIIA interaction. No significant interaction with the {gamma}-subunit was observed, which reinforces the conclusion that the {alpha}-subunit is mainly required for contacting CREM. Our deletion analysis revealed that, although the first 127 aa of TFIIA appear to be essential, there is evidence of a unique domain within TFIIA{alpha} for interaction with CREM.

There is clear evidence of coordinated expression of TFIIA and CREM in testis. First, TFIIA and CREM transcript levels are developmentally regulated in a similar manner (Fig. 5BGo). Secondly, both TFIIA and CREM are overexpressed in the testis (Fig. 5AGo), being predominantly localized in germ cells, namely spermatocytes and spermatids, (Fig. 5DGo). In addition, the levels of TFIIA seem to be modulated during the spermatogenic wave (Fig. 6AGo), with an increase at stages V–IX where also CREM protein levels peak. Finally, our immunofluorescence analyses demonstrate that the two proteins are localized in nuclei of the same germ cell types within the seminiferous tubules (Figs. 6BGo and 7Go). Indeed, during the spermatogenic wave both CREM and TFIIA appear to follow similar overlapping expression profiles.

Our understanding of the relationship between testis gene expression and the general transcription factors is still incomplete. Elevated levels of expression of testis-specific activators, such as CREM, seem to be accompanied by high expression of factors belonging to the basal transcriptional machinery, suggesting that general transcription factors and their homologs may have a unique role in testis gene regulation. In addition, it is apparent that many basal transcription factors have unique expression patterns during testis development (e.g. TBP, TLF, Drosophila TAFIIs, TFIIA, ALF, TFIIE, the large subunit of Pol II, TAF7L) (19, 24, 25). Thus, testis-specific gene expression may require, other than specialized transcription factors, the concerted action of specialized coactivators and global/general regulators.

Evidence of tissue-specific TFIIA homologs has been provided. TFIIA{tau}/ALF is highly expressed in testis and it is believed to be a functional tissue-specific counterpart of TFIIA (26, 27). This gene shares a significant sequence similarity with the evolutionarily conserved amino- and carboxyl-terminal domains of TFIIA{alpha}ß; its product, together with the {gamma}-subunit, forms a complex able to support basal and activated transcription in reactions reconstituted with TFIIA-depleted nuclear extracts. In spite of the high homology shared by TFIIA and ALF, no significant interaction between ALF and CREM was found, thus confirming the high specificity of CREM-TFIIA association. This also suggests that there is no redundancy between the two homologs, at least with respect to the interaction with CREM. Thus, what could be the functional significance of such specificity? It has been proposed that ALF may be required in vivo for the expression of a subset of class II testis-specific genes or for helping to mediate the function of testis-specific transcriptional factors. In this scenario, it could be speculated that the different affinity of CREM for TFIIA and ALF, respectively, may reflect the fact that CREM would utilize TFIIA to activate the expression of given subsets of testis-specific genes, whereas other testis-specific activators would recruit ALF in the assembly of distinct preinitiation complexes to transcribe different genes (see Fig. 8Go). TFIIA has also been shown to interact with TLF (1) and this association could recruit the complex CREM/TFIIA to TATA-less promoters (see model in Fig. 8Go). Indeed, several testis-specific promoters, including some CREM-regulated genes, do not contain a canonical TATA element (21). It is also notable that mutations in the Drosophila trf gene and ablation of the tlf gene in mice result in impaired spermatogenesis and male sterility (19, 34, 35), paralleling the phenotype observed for the CREM-null mice (13). It is conceivable that a physiologically complex process of tightly coordinated events such as spermatogenesis not only requires high levels of transcription, but also a strict control of the activity of both specific and general transcription factors overexpressed in testis.



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  		Fig. 8. Proposed Model for CREM-Mediated Transcription in Testis

In male germ cells, CREM associates with its specific coactivator ACT which, in turn, is bound by the kinesin Kif17b that regulates ACT nucleus-cytoplasm shuttling. In this model, transcriptional activation involves contacts of CREM with components of the basal transcriptional machinery, and in particular with TFIIA. TFIIA can associate with both TBP (TATA-binding protein) and TLF (TBP-like factor). Association with TLF could help support CREM-mediated transcription from TATA-less promoters. TFIIA may contact other components of the basal transcription machinery (arrow with question mark), such as testis-specific TAFs.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Yeast Plasmid Construction
pGal4-DBD-CREM, pGal4-DBD-CREM Ala117, pGal4-DBD-CREB, pGal4-DBD-Sp1, and pGal4-DBD-c-Jun plasmids have been described (16, 43). TFIIA{gamma} and ALF cDNAs (encoding their complete ORFs) were obtained by PCR from a mouse testis cDNA library and cloned into pGAD424 vector (CLONTECH, Palo Alto, CA). Expression vectors for CREM functional domains fused to the Gal4-DBD have also been described (15). TFIIA{alpha} deletions were generated by the use of appropriate restriction enzymes in the context of the pGAD424 vector.

Yeast Analysis
A murine adult testis cDNA library (CLONTECH) was cotransformed with the pGBT-CREM bait plasmid in CG1945 yeast cells. Yeast two-hybrid screening was performed as described (15). TFIIA was the second most frequent clone found in the screen after ACT. Yeast transformation and ß-galactosidase assay were carried out in Y190 yeast strain, as described in the CLONTECH Matchmaker Two-Hybrid System Protocol. ß-Galactosidase activity was calculated in Miller units and results are means of two to four independent experiments, each one done in triplicate. Equal expression levels of the different Gal4 fusion proteins were checked with anti-Gal4DBD and anti-Gal4AD monoclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) by Western blot analysis as described (16).

RNA Analysis
Total RNA was extracted from mouse tissues as previously described (44) and analyzed by ribonuclease protection (45). [{alpha}-32Uridine triphosphate] antisense riboprobes were generated using an in vitro transcription kit (Promega). The TFIIA riboprobe corresponds to nt +1/+158 of the mouse TFIIA{alpha}/ß cDNA/9H clone. ACT and CREM riboprobes were as described (16, 46). In all ribonuclease protection analyses, tRNA was used as a control for nonspecific protection. A mouse ß-actin riboprobe was used as internal control to monitor the loading of equal amounts of RNA (fragment from nt +193 to nt +331 of the mouse cDNA).

Preparation of TFIIA Antiserum and Protein Analyses
Rabbit anti-TFIIA antiserum was prepared by sequential immunization with 100 µg of a peptide ({alpha}293) spanning aa 255–273 of TFIIA protein, coupled to keyhole limpet hemocyanin (from Pierce, Rockford, IL). TFIIA antibodies were purified from serum by using an affinity resin containing the immunization peptide immobilized onto a Sulfolink coupling gel (from Pierce), according to manufacturer’s instructions. Mouse Sertoli, spermatocytes and spermatid cells were separated by centrifugation as described (18). Testis and purified germ cell protein extracts for Western blot analysis were prepared as previously described (28). Purified TFIIA antibodies were used at a dilution of 1:2000. Immunocomplexes were detected by enhanced chemiluminescence (Pierce).

Coimmunoprecipitation Assays
The TFIIA expression vector pCS2Myc-TFIIA was constructed by inserting TFIIA{alpha} ORF (nt +1 to nt +822) into the pCS2Myc plasmid (47). COS cells were transfected with 10 µg of each plasmid and harvested after 48 h in 1 ml of EBC [50 mM Tris-HCl (pH 8.0), 170 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF] containing 1 mM phenylmethylsulfonyl fluoride and 10 µg/ml of aprotinin and leupeptin. Lysates were incubated at 4 C for 3 h with purified polyclonal antibodies raised against CREM. Beads were washed four times in NETN [10 mM Tris-HCl (pH 8.0), 250 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40]. CREM antibodies used for immunoprecipitation were described (15). Immunocomplexes were revealed by Western blot analysis using anti-Myc (9E12) monoclonal antibodies.

Immunohistochemistry
Rat seminiferous tubule segments at defined stages were isolated for Western analysis using the transillumination-assisted microdissection method (33) and pooled to obtain 4–5 cm, equivalent to 4–5 mg of wet weight tissue. Protein extracts were made by shearing the tubule segments in 2x boiling Laemmli buffer containing 10 mM ß-mercaptoethanol. The extracts were analyzed by SDS-PAGE followed by staining with Comassie Blue to normalize each preparation. Immunoblotting and chemiluminescence detection were performed by standard methods. Immunohistochemistry was performed on fixed sectioned seminiferous tubules from squash preparations of microdissected tubules as previously described (13, 19), using the described CREM antibodies (15) and the TFIIA {alpha}293 antibodies described above.


   ACKNOWLEDGMENTS
 
We thank Lucia Monaco, Irwin Davidson, and Betina Macho for help and discussions; and Stéphanie Roux and Estelle Heitz for expert technical help.


   FOOTNOTES
 
This work was supported by Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Centre Hospitalier Universitaire Régional, Fondation de la Recherche Médicale, Université Louis Pasteur, Human Frontier Science Program (RG-240), Organon (Akzo/Nobel), Electricité de France and Association pour la Recherche sur le Cancer.

D.D.C. and G.M.F. contributed equally to this work.

Present address for D.D.C.: Institute of Genetics and Biophysics "A. Buzzati-Traverso," Via G. Marconi 12, 80125 Naples, Italy.

Abbreviations: aa, Amino acids; ACT, activator of CREM in testis; AD, activation domain; CBP, CREB binding protein; CRE, cAMP-responsive element; CREB, cAMP response element binding protein; CREM, CRE modulator; KID, kinase-inducible domain; ORF, open reading frame; PS, pachytene spermatocytes; RS, round spermatids; TBP, TATA-binding protein; TF, transcription factor; TLF, TBP-related factor.

Received for publication July 17, 2003. Accepted for publication September 18, 2003.


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