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Genes to Cells (2004) 9, 1125-1135. doi:10.1111/j.1365-2443.2004.00795.x
© 2004 Blackwell Publishing or its licensors
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GRIP1{tau}, a novel PDZ domain-containing transcriptional activator, cooperates with the testis-specific transcription elongation factor SII-T1

Akihito Nakata, Takahiro Ito, Makiko Nagata, Sayaka Hori and Kazuhisa Sekimizu*

Department of Developmental Biochemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
SII-T1 is a tissue-specific member of the transcription elongation factor S-II that is expressed specifically in male germ cells. In the present study, we have identified a protein named GRIP1{tau} interacting with SII-T1 by yeast two-hybrid screening. GRIP1{tau} is a novel isoform of glutamate receptor-interacting protein 1 (GRIP1) that associates with the cytoplasmic domain of the {alpha}-amino-3-hydroxy-5-methyl-4-isoaxazolepropionate (AMPA)-type glutamate receptor. GRIP1{tau} is a testis-specific nuclear protein that activates transcription when fused with a GAL4 DNA binding domain in GAL4-responsive reporter gene assays. The transactivation domain of GRIP1{tau} overlapped with the region essential for interaction with SII-T1, as revealed by co-immunoprecipitation assays. Also, transactivation by GRIP1{tau} was stimulated by SII-T1 in a dose-dependent manner. Therefore, we propose that GRIP1{tau} is a novel testis-specific transcriptional activator regulated by interaction with the testis-specific transcription elongation factor SII-T1.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Spermatogenesis is a highly organized and specialized process that produces functional sperm with a haploid genome. The developmental process includes meiosis and drastic changes in the cell morphology, which are regulated by many genes expressed spatio-temporally in testis (Eddy 2002). Transcription factors with sequence-specific DNA binding activity direct the spatio-temporal regulation of such genes and some of them are essential for spermatogenesis (Toscani et al. 1997; Martianov et al. 2002). For example, CREM{tau} binds cAMP-responsive elements (CREs) and regulates a set of genes with CRE in their promoters, which is required for the development of the spermatid as revealed by targeted gene disruption in mice (Nantel et al. 1996; Sassone-Corsi 1998).

Regulation of the gene expression during the transcription elongation steps in spermatogenesis is poorly understood. Among the transcription elongation factors identified to date, S-II, ELL and Elongin have testis-specific paralogues, SII-T1, ELL3 and ElonginA2 (Xu et al. 1994; Ito et al. 1996; Aso et al. 2000; Miller et al. 2000). The mechanisms by which these elongation factors regulate gene expression during spermatogenesis, however, remain unclear.

S-II promotes cleavage of the nascent transcript by the RNA polymerase II arrested on the template DNA during the elongation step and is involved in the stimulation of transcription elongation and maintenance of the transcriptional fidelity in eukaryotes (Reines et al. 1992; Thomas et al. 1998; Wind & Reines 2000; Koyama et al. 2003). In mammals, there are three S-II-related proteins, S-II, SII-T1 and SII-K1, each of which exhibits distinct tissue distributions (Xu et al. 1994; Ito et al. 1996; Taira et al. 1998). At their C-terminal domains, all three paralogues share a highly conserved region essential for binding with the RNA polymerase II core enzyme, and this region is also required for the stimulation of transcription elongation by RNA polymerase II (Nakanishi et al. 1995; Shimoaraiso et al. 1997). The N-terminal region is involved in the association with the RNA polymerase II holoenzyme (Pan et al. 1997), while the intervening region is distinct among the paralogues.

In this study, we screened factors interacting with the N-terminal and the intervening domains of SII-T1 using a yeast two-hybrid system, and identified a testis-specific variant of glutamate receptor-interacting protein 1 (GRIP1), named GRIP1{tau}. GRIP1 is a PDZ domain-containing protein that specifically binds to the C termini of {alpha}-amino-3-hydroxy-5-methyl-4-isoaxazolepropionate (AMPA) glutamate receptor subunits and is thought to be involved in the synaptic targeting of these receptors in neurones (Dong et al. 1997). In contrast, GRIP1{tau} distributes in the nucleus and contains a transcriptional activation domain, and its transactivating potential is stimulated by the interaction with transcription elongation factor SII-T1.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Identification of SII-T1 interacting protein through a yeast two-hybrid screen

To identify the factors interacting with the male germ cell-specific transcription elongation factor SII-T1, we performed a GAL4-based yeast two-hybrid screen against a mouse testis cDNA library using the N-terminal 180-amino acid region of mouse SII-T1 as bait and isolated 34 positive clones from three million clones. Sequencing results of the positive clones revealed that 24 clones were derived from the PDZ domain-containing protein GRIP1 and all the 24 clones were derived from the identical GRIP1 mRNA isoform because the nucleotide sequences of these clones were overlapping one another. Of the 24 GRIP1 clones, 22 clones were not fused in-frame with the GAL4 transcriptional activation domain of the prey vector pGAD10. Because the results described below revealed that these GRIP1 clones encode a novel isoform of GRIP1 with testis-specific expression, we named the protein GRIP1{tau}.

GRIP1{tau} is a novel isoform of GRIP1

BLAST searches against the GENBANK database using the consensus cDNA sequences of the GRIP1 clones as query revealed that it is the same as those of the RIKEN full-length cDNA clones deposited under the accession numbers AK029905 and AK016420. These two cDNAs contained open-reading frames encoding the same protein composed of 632 amino acid residues with a predicted molecular mass of 69 kDa, and contained four PDZ domains. These cDNAs contain a region highly homologous with the GRIP1a-L cDNA (accession number AB051561), which encodes a protein interacting with AMPA-type glutamate receptor subunits (Yamazaki et al. 2001). Alignment of the putative amino acid sequences revealed that GRIP1{tau} protein encoded by the AK029905 was identical to the C-terminal half of GRIP1a-L protein, except for in the following three aspects: (i) N-terminal 36 amino acid residues of GRIP1{tau} are not included in the GRIP1a-L protein (red letters in Fig. 1); (ii) two regions encoding amino acid residues 756–820 and 911–925, that are located between PDZ domains 6 and 7 of the GRIP1a-L, are absent in the GRIP1{tau} (indicated as deletion 1 and deletion 2 in Fig. 1).



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  		Figure 1  GRIP1{tau} is a novel isoform of GRIP1. The putative amino acid sequence of GRIP1{tau} (top lines) is aligned with that of GRIP1a-L (bottom lines). Dashes indicate gaps introduced to maximize similarity. PDZ domains are underlined with their relative positions in GRIP1a-L (PDZ1-7). Red letters denote the region specific for GRIP1{tau} (amino acid residues 1–36). Amino acids 756–820 and 911–925 of GRIP1a-L are not present in GRIP1{tau} (indicated as deletion 1 and deletion 2, respectively). Asterisks indicate identical amino acid residues.

 
The results described above suggested that GRIP1{tau} and GRIP1a-L transcripts are splicing variants of the GRIP1 gene. To confirm this notion, we analysed the genomic structure of the gene. We identified the GRIP1 gene in the mouse genome dataset of the Celera Discovery System (CDS) by a BLAST search using the nucleotide sequence of the GRIP1{tau} cDNA for the query (CDS ID# mCG19224. The GRIP1 gene spans over 250 kb and maps to chromosome 10. The nucleotide sequence alignments of the GRIP1 genomic DNA and the two cDNAs indicated that GRIP1a-L consists of 25 exons and GRIP1{tau} is composed of 13 exons (Table 1). All introns contain splice acceptor and donor consensus sequences, GT and AG, respectively, at their boundaries. The two regions missing in the GRIP1{tau} cDNA (deletions 1 and 2 in Fig. 1) were derived from exons 20 and 23, respectively. These results indicated that GRIP1{tau} and GRIP1a-L were transcript variants transcribed from the same GRIP1 gene.


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  		Table 1  Exon/intron structure of the murine GRIP1 gene
 
Germ cell-specific expression of GRIP1{tau} in the testes

We identified GRIP1{tau} as an interacting protein of testis-specific SII-T1. GENBANK database searches revealed that cDNA clones for GRIP1{tau} were isolated from testis cDNA libraries. These results led us to hypothesize that GRIP1{tau} is a testis-specific isoform, while GRIP1a-L is expressed in the brain and testis (Dong et al. 1997). To examine the expression of GRIP1{tau} protein in testis, we performed Western blot analysis of the testis and brain extracts with the monoclonal antibody directed against the C-terminal region of GRIP1a-L (Fig. 2A,B). We observed a testis-specific protein with a molecular mass of 70 kDa that was in good agreement with the theoretical molecular size of GRIP1{tau} protein (69 kDa). This species was not detected in the brain extract, suggesting that GRIP1{tau} is expressed specifically in the testis. To confirm the testis-specific expression of GRIP1{tau}, we analysed its mRNA expression in various mouse tissues by reverse transcription-polymerase chain reaction (RT-PCR) using a pair of primers specific for GRIP1{tau} (primers a and b in Fig. 2A) among GRIP1 mRNA isoforms. As shown in Fig. 2(C), we observed a 745-bp amplification product in testis but not in brain, liver, heart, kidney and spleen. From these results, we concluded that GRIP1{tau} is expressed specifically in testis.



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  		Figure 2  GRIP1{tau} is specifically expressed in the germ cells of the testis. (A) Schematic illustrations of the primary structures of GRIP1{tau} and GRIP1a-L. Dashed lines indicate the corresponding regions in the two GRIP1 isoforms. Shaded boxes indicate PDZ domains and the hatched box indicates GRIP1{tau}-specific region. The bar indicates the region recognized by the GRIP1 antibody used in (B). Arrows labelled a, b, c and d indicate the primers used in the RT-PCR analyses in (C) and (D). (B) Western blot analysis of mouse testis and brain. Testis and brain extracts were analysed by immunoblotting with anti-GRIP1 monoclonal antibody. Filled and open arrowheads indicate GRIP1a-L and GRIP1{tau} proteins, respectively. (C) Testis-specific expression of GRIP1{tau} mRNA by RT-PCR analysis. A pair of primers (a and b) specific for GRIP1{tau} among GRIP1 isoforms was used. A PCR product of 745 bp was detected in the testis, but not in brain, liver, heart, kidney and spleen. The bottom panel indicates the equivalent amplification of hypoxanthine-guanine phosphorybosyl transferase transcripts. M, 100-bp DNA ladder marker. (D) RT-PCR analyses of GRIP1 isoforms in wild-type and W/Wv mutant testis. (i) A pair of primers (a and b) specifically amplifies GRIP1{tau} transcript. (ii) A pair of primers (c and d) amplifies both isoforms, but differ in the sizes of the amplicons (755 bp for GRIP1{tau} and 995 bp for GRIP1a-L) due to deletions in the region between the last two PDZ domains of GRIP1{tau}. (iii) The bottom panel shows the equivalent amplification of hypoxanthine-guanine phosphorybosyl transferase transcripts. RT, the control reaction without reverse transcription. M, 100-bp DNA ladder marker.

 
In testis, there are two types of cells: the germ cells that undergo spermatogenesis, and somatic cells that support the germ cells. The testis of the Whitespotting mutant mouse (W/Wv) contain somatic cells alone because of a mutation in the c-kit gene that is essential for differentiation of the germ cells (Manova et al. 1990; Ohta et al. 2003). To determine whether GRIP1{tau} is expressed exclusively in the male germ cells, we analysed GRIP1{tau} mRNA expression in the W/Wv mutant mouse testes. We performed RT-PCR analyses with two pairs of primers (Fig. 2D). One pair of primers a and b specifically amplified the GRIP1{tau} transcript, and the other pair (primers c and d) amplified both GRIP1{tau} and GRIP1a-L, but in the latter reaction the sizes of the amplicon differed for these transcripts because of the two deletions in the GRIP1{tau} transcript. When primers a and b were used, we observed a 745-bp amplification product from wild-type testis, but not from the W/Wv mutant mouse testis (Fig. 2D, top panel). With primers c and d, we observed two signals, 995-bp (GRIP1a-L) and 755-bp (GRIP1{tau}) in the wild-type, while in the mutant we only detected a 995-bp RT-PCR product (Fig. 2D, middle panel). These results indicated that GRIP1{tau} mRNA expression is restricted to the spermatogenic cells. We previously demonstrated that SII-T1 expression is not detectable in the W/Wv mutant mouse testis and is expressed specifically in spermatocytes (Ito et al. 1996). Therefore, we concluded that both GRIP1{tau} and SII-T1 are male germ cell-specific factors.

Nuclear distribution of GRIP1{tau}

GRIP1a-L protein associates with the C-terminal domain of AMPA glutamate receptor subunits and functions in the cytoplasm (Dong et al. 1999). The present study indicated that GRIP1{tau} binds with the transcription elongation factor, which is localized to the nucleus. To analyse if GRIP1{tau} is in the nucleus, we transfected COS7 cells with the plasmid DNA encoding FLAG-tagged GRIP1{tau}, and analysed its intracellular distribution by indirect immunofluorescent staining using anti-FLAG antibody. FLAG-GRIP1{tau} localized to the nucleus (Fig. 3A, panel i). In some cells, both the nucleus and cytoplasm were stained (Fig. 3A, panel iv), suggesting that GRIP1{tau} could distribute in the cytoplasm. To confirm the nuclear distribution of GRIP1{tau} in the testicular cells, we performed Western blot analysis against the nuclear and cytoplasmic fractions prepared from mouse testicular cells. Histone and ß-tubulin proteins were enriched in the nuclear and cytoplasmic fractions, respectively (Fig. 3B, middle and bottom panels). Under these conditions, a significant amount of the GRIP1{tau} protein was recovered in the nuclear fractions (Fig. 3B, open arrowhead). Some of the GRIP1{tau} protein was recovered in the cytoplasmic fraction, consistent with the immunofluorescence analysis. In contrast, most of the GRIP1a-L protein was recovered in the cytoplasmic fraction (Fig. 3B, closed arrowhead). These findings indicated that GRIP1{tau} is in the nucleus of the spermatogenic cells.



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  		Figure 3  Nuclear distribution of GRIP1{tau}. (A) COS-7 cells were transiently co-transfected with the vectors expressing FLAG-tagged GRIP1{tau} and Xpress-tagged SII-T1, then processed for immunofluorescence analyses using rabbit anti-FLAG antibody and Alexa 568-conjugated anti-rabbit immunoglobulin antibody for GRIP1{tau} (i, iv), and anti-Xpress monoclonal antibody and Alexa 488-conjugated anti-mouse immunoglobulin antibody for SII-T1 (ii, v). DAPI was used to stain nuclei (iii, vi). (B) Western blot analysis of nuclear and cytoplasmic proteins from mouse testis. Protein extract from whole testis (Testis Lysate), nuclear and cytoplasmic proteins were separated by SDS–PAGE and analysed by immunoblotting with the indicated antibodies.

 
Region encoding amino acids 340–507 of the GRIP1{tau} is required for the interaction with SII-T1

To assess the interaction of GRIP1{tau} and SII-T1 under physiologic conditions, we analysed whether these factors co-immunoprecipitate from the cell extract. We introduced constructs expressing Xpress-tagged SII-T1 and FLAG-tagged GRIP1{tau} into COS7 cells and performed immunoprecipitation with anti-FLAG antibody followed by Western blot analysis with anti-Xpress antibody. We detected Xpress-SII-T1 protein in the immunoprecipitate fraction (Fig. 4B, lane 2). We did not observe the Xpress-SII-T1 signal when we introduced the FLAG vector without GRIP1{tau} cDNA (Fig. 4B, lane 1). We did not observe co-immunoprecipitation of S-II, suggesting that GRIP1{tau} specifically interacts with SII-T1 among S-II family proteins (data not shown). Next we performed co-immunoprecipitation assays of the GRIP1{tau} deletion mutants (Fig. 4A) to determine the region required for interaction with SII-T1. Xpress-SII-T1 was co-immunoprecipitated with the FLAG-GRIP{Delta}1 (Fig. 4B, lane 3), but not with the FLAG-GRIP{Delta}2 or {Delta}3 (Lanes 4 and 5), indicating that the region encoding amino acids 340–507 was essential for the interaction (region indicated by the black bar in Fig. 4A).



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  		Figure 4  Identification of the region essential for the interaction with SII-T1. (A) Schematic illustrations of the FLAG-tagged GRIP1{tau} and its deletion mutants. Shaded boxes indicate PDZ domains and a hatched box indicates GRIP1{tau}-specific region. The bar indicates the region (residues 340–507) required for the interaction with SII-T1. (B) Co-immunoprecipitations of SII-T1 by GRIP1{tau} or its derivatives. COS7 cells transiently expressing Xpress-tagged SII-T1 and FLAG-tagged GRIP1{tau} or its deletion mutants were lysed, and the crude lysates were used for immunoprecipitation with anti-FLAG antibody. The lysates (INPUT) and immunoprecipitates (IP) were subjected to SDS–PAGE and analysed by immunoblotting with the anti-FLAG antibody (top panel) or anti-Xpress antibody (middle and bottom panels). Transfection with FLAG-empty vector that does not harbour the GRIP1{tau} cDNA sequence acted as a negative control.

 
GRIP1{tau} possesses a transactivation domain

In the two-hybrid screen, we isolated many GRIP1{tau} clones that were not fused in-frame with the GAL4 transcriptional activation domain encoded in the two-hybrid prey vector. Therefore, we hypothesized that GRIP1{tau} itself functions as a transcriptional activator. To test this hypothesis, we analysed the transactivation potential of GRIP1{tau} protein. We introduced a construct encoding GAL4DBD-fused GRIP1{tau} along with a luciferase reporter construct driven by the GAL4 binding cis-element (GAL4-responsive upstream activating sequence) into the STO mouse embryonic fibroblast cell line and assayed luciferase activity in the cell extract. GAL4DBD-fused GRIP1{tau} activated luciferase expression in a dose-dependent manner (Fig. 5B), indicating that GRIP1{tau} contains a transcriptional activation domain. Next, we examined transactivation by the GRIP1{tau} deletion mutants to determine the transactivation domain (Fig. 5A). Full-length GRIP1{tau} and GRIP{Delta}1 activated luciferase expression 14- and 6-fold, respectively (Fig. 5C). Transactivation by GRIP{Delta}2 and {Delta}3 deletion mutants, however, was not detected, suggesting that the region of amino acid residues 340–507 was required for the transcriptional activation (indicated by the black bar in Fig. 5A). This was the same region essential for the interaction with SII-T1, suggesting that transcriptional activation by GRIP1{tau} is regulated by the interaction with SII-T1.



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  		Figure 5  GRIP1{tau} has intrinsic transactivation potential. (A) Schematic representation of effector constructs used in the experiment. Shaded boxes indicate PDZ domains and a hatched box indicates GRIP1{tau}-specific region. GAL4-DBD, DNA binding domain from yeast transcription factor GAL4. The bar indicates the region required for the transcriptional activation. (B) Dose-dependent activation of GAL4-responsive firefly luciferase reporter by GRIP1{tau} fused with GAL4 DBD. Values on the horizontal axis were the amounts of the GAL4DBD-GRIP1{tau} vector used. Relative luciferase activity (Relative activation) induced by GAL4DBD-GRIP1{tau} construct was calculated by defining the luciferase activity in the presence of GAL4DBD alone (GAL4DBD-empty vector) as 1.0. Error bars represent the standard deviations of two independent experiments. (C) Region encoding residues 340–507 is essential for the transactivation domain. Relative luciferase activities were defined as in (B). Error bars represent the standard deviations of two independent experiments.

 
SII-T1 enhances transcriptional activation by GRIP1{tau}

To examine whether SII-T1 affected transcriptional activation by GRIP1{tau}, we analysed the effect of co-introduction of the SII-T1 expression vector on the transactivation by GRIP1{tau}. When we introduced GAL4DBD-GRIP1{tau}, co-introduction of a vector expressing FLAG-SII-T1 stimulated the transcriptional activation by GRIP1{tau} in a dose-dependent manner (Fig. 6B). In contrast, there was no enhancement by SII-T1 when the construct expressed GAL4DBD alone (Fig. 6A). These results suggested that SII-T1 functions to enhance the transactivation by GRIP1{tau}.



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  		Figure 6  SII-T1 stimulation of the GRIP1{tau} transcriptional activity. A vector expressing GAL4DBD alone (A) or GAL4DBD-GRIP1{tau} (B) was transfected along with the increasing doses of FLAG-tagged SII-T1 expression vector (amounts indicated on the horizontal axis), and luciferase reporter activation were analysed. Relative activations of the luciferase expressions were calculated by defining the luciferase activity by GAL4DBD alone in the absence of FLAG-SII-T1 as 1.0 (indicated as (–)).

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
GRIP1 protein associates with AMPA glutamate receptor subunits GluR2 and GluR3 in brain neuronal cells and is involved in trafficking these receptors to the plasma membrane and the formation of the clusters of AMPA receptors on the membrane (Dong et al. 1997; Hirai 2001). GRIP1 mRNA is expressed in testis, but its function remains unclear, except for a report that GRIP1 interacts with DNA polymerase ß (Jonason et al. 2001). The present study indicates that a novel testis-specific isoform of GRIP1 is a transcriptional activator. Therefore, we propose a new aspect of GRIP1 function.

Structural characteristics of GRIP1{tau}

GRIP1{tau} protein has four PDZ domains homologous to those of GRIP1a-L, except that there are two deleted regions between the last two PDZ domains when compared with GRIP1a-L protein. Analysis of the GRIP1 gene structure revealed that these two deletions are as a result of exon skipping of exons 20 and 23, respectively. The N-terminal 36-amino acid residues of GRIP1{tau} are specific to GRIP1{tau} and are encoded in exon 12, which is skipped in GRIP1a-L mRNA. A dbEST database search with the nucleotide sequence of the GRIP1{tau}-specific region used for the query revealed that the cDNA clones containing the sequence were isolated only from the testis cDNA library (data not shown). Therefore, the GRIP1 gene might contain an alternative promoter that directs testis-specific expression and the GRIP1{tau} transcript might be expressed from the promoter.

Yamazaki et al. (2001) reported multiple splicing variants of the GRIP1 gene. They identified three variants with an insertion/deletion when compared with the GRIP1a-L transcript. These three regions of insertion/deletion seem to correspond to exons 2, 10, and 23. Yu et al. (2001) reported a brain-specific isoform, GRIP1b, that contains five PDZ domains. Their and our results indicate that GRIP1 protein has several isoforms with smaller molecular sizes that are expressed in the brain and/or testis. This is consistent with our immunoblot analysis results with anti-GRIP1 antibody (Fig. 2B) in which we observed multiple immunoreactive species that might be derived from the transcript variants.

Western blot analysis indicated that the largest GRIP1 species in the testis extract migrated slower than GRIP1a-L in the brain (Fig. 2B). Because cysteine residues in the N-terminal region of GRIP1b-L could be palmitoylated (Yamazaki et al. 2001), it is possible that the largest GRIP1 species in the testis is also modified by N-terminal palmitoylation.

Nuclear distribution of GRIP1{tau}

We have shown the GRIP1{tau} exists both in the nucleus and cytoplasm, while most of the GRIP1a-L protein is located in the cytoplasm (Fig. 3B). Dong et al. reported that GRIP1 protein forms homo- and hetero multimers through their PDZ domains 4–6 (Dong et al. 1999), which correspond to the first three PDZ domains of GRIP1{tau} and are conserved between GRIP1a-L and GRIP1{tau}. We speculate that the GRIP1a-L and GRIP1{tau} complexes are located on the plasma membranes of the testicular germ cells and GRIP1{tau} translocates to the nucleus upon stimulation by glutamate and activates transcription of the genes involved in sperm differentiation. Some of GRIP1a-L proteins, although the amount was small, were present in the nucleus (Fig. 3B). As GRIP1a-L contains a region highly homologous to the transcriptional activation domain of GRIP1{tau}, GRIP1a-L may function as a transcriptional activator.

SII-T1 interaction with GRIP1{tau}, and its functional relevance

We have shown that the region encoding amino acid residues 340–507 that is located between the last two PDZ domains of GRIP1{tau} is essential for the interaction with SII-T1. This domain is also required for the transcriptional activation by GRIP1{tau}, suggesting that SII-T1 binds to GRIP1{tau} via its transactivation domain. SII-T1 interacts with GRIP1{tau} through its N-terminal half because we used the 1–180-amino acid residue region of SII-T1 in the two-hybrid screen to isolate GRIP1{tau} clones. A recent report describing the crystal structure of yeast RNA polymerase II and S-II suggests that the C-terminal half of S-II is buried in the RNA polymerase II core enzyme, while the N-terminal half is exposed outside the polymerase (Kettenberger et al. 2003). We previously demonstrated that S-II interacts with a novel transcriptional activator FESTA through its N-terminal region (Saso et al. 2003). Therefore, we propose that the N-terminal region of S-II proteins is involved in binding with the transactivators.

Because the region required for interaction with SII-T1 and transactivation by GRIP1{tau} overlaps (Figs 4 and 5), we examined whether SII-T1 enhances the transactivation potential of GRIP1{tau}. Introduction of the SII-T1 expression vector stimulated the activation of luciferase expression by GAL4DBD-GRIP1{tau} (Fig. 6). These results suggested that SII-T1 regulates the transcriptional activation by GRIP1{tau} through the physical interaction. It remains to be elucidated whether the regulation of the GRIP1{tau} transcriptional activation by SII-T1 interaction has a role in spermatogenesis.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Yeast two-hybrid screen

A cDNA fragment encompassing amino acid residues 1 through 180 of mouse SII-T1 was amplified by polymerase chain reaction (PCR) using mouse SII-T1 cDNA as a template, and inserted in-frame into the SmaI site of pAS2-1 (Clontech, Palo Alto, CA). The resulting construct, pAS2-1/TL1-180, was used as bait in a two-hybrid screen and was co-transfected into the Saccharomyces cerevisiae AH109 strain with a mouse testis cDNA library based on the pGAD10 vector (Clontech). Two-hybrid screening and ß-galactosidase assays were performed essentially as previously described (Saso et al. 2003). The cDNA sequences of the positive clones were analysed using an ABI PRISM 377XL DNA sequencer (PE Applied Biosystems, Foster City, CA).

Identification and structure analyses of the GRIP1 gene

Nucleotide sequences of the cDNA encoding GRIP1{tau} (accession no. AK029905) and GRIP1a-L (accession no. AB051561) were retrieved from the GENBANK Database. The genomic sequence for the GRIP1 gene was obtained through the Celera Discovery System (http://www.celeradiscoverysystem.com/) by a nucleotide sequence similarity search using the cDNA sequence for the query. We defined the 5'-end of each cDNA clone as the transcription start site (+1 site) and exons were deduced from the nucleotide sequence alignment of the genomic DNA and cDNA.

Western blot analysis of GRIP1 protein isoforms

Mouse tissues were homogenized in 2 mL of phosphate-buffered saline with a Teflon-glass homogenizer. The homogenate was centrifuged at 15 000 g for 5 min at 4 °C and the supernatant was used as an extract from mouse testes for immunoblotting. Protein concentrations were determined using the Lowry method with bovine serum albumin as a standard (Lowry et al. 1951). Extracts containing 50 µg of protein were analysed by sodium dodecyl sulphate—polyacrylamide gel electrophoresis (SDS–PAGE) and transferred on to immobilon-P polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocking, the blots were probed with 250 ng/mL anti-GRIP1 monoclonal antibody (BD Transduction, San Diego, CA) at 4 °C overnight, followed by incubation with a 1 : 5000 dilution of anti-mouse IgG horseradish peroxidase-linked species-specific whole antibody (Amersham Biosciences, Piscataway, NJ). The immunoreactive proteins were visualized by Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer, Boston, MA). Preparations of nuclear and cytoplasmic extracts of mouse testes were performed essentially as previously described (Kashiwabara et al. 2000).

Reverse transcription-polymerase chain reaction (RT-PCR) analyses of GRIP1{tau} mRNA

RNA extraction and RT-PCR analyses were performed essentially as previously described (Ito et al. 1996). PCR primers used were as follows: primer a, 5'-ATGACAGCAAAACGAGCT-3'; primer b, 5'-CTCTTGCTCGTCTGAGTTATC-3'; primer c, 5'-CACGGTGGAGCTGAAGCGC-3'; primer d, 5'-ATGTAGCTCCACCGGAGT-3'. The primer pair used for hypoxanthine-guanine phosphorybosyl transferase (HPRT) transcript amplification were as follows: sense, 5'-CACTGCTTTCCGGAGCGGTA-3'; anti-sense, 5'-GTTGAGAGATCATCTCCACC-3'. PCR conditions for GRIP1 transcripts were as follows: PCR with primers a and b, 94 °C 2 min + (94 °C 1 min, 46 °C 1 min, 72 °C 1 min) x 35 cycles + 72 °C 5 min; PCR with primers c and d, 94 °C 2 min + (94 °C 1 min, 55 °C 1 min, 72 °C 1 min) x 35 cycles + 72 °C 5 min; PCR with HPRT primers, 94 °C 2 min + (94 °C 1 min, 60 °C 1 min, 72 °C 1 min) x 35 cycles + 72 °C 5 min. Reaction products were analysed by agarose gel electrophoresis, and visualized by ethidium bromide staining.

Expression vectors

Expression vectors of FLAG-tagged full-length GRIP1{tau} (amino acids residues 1–632), GRIP{Delta}1 (residues 1–507), GRIP{Delta}2 (residues 1–339), and GRIP{Delta}3 (residues 1–249) were constructed by inserting the corresponding cDNA fragments into the EcoRV site of the pFLAG vector. Expression vectors of the GAL4 DNA binding domain (GAL4DBD)-fused GRIP1{tau} derivatives, namely, GAL4DBD-GRIP1{tau} (residues 1–632), GAL4DBD-GRIP{Delta}1 (residues 1–507), GAL4-GRIP{Delta}2 (residues 1–339) and GAL4DBD-GRIP{Delta}3 (residues 1–249) were constructed by inserting the corresponding cDNA fragments into the EcoRV site of pBIND (Promega, Madison, WI). The expression vector of FLAG-tagged and Xpress-tagged SII-T1 was constructed by inserting the cDNA encoding the entire open-reading frame of mouse SII-T1 into the EcoRV site of pFLAG and pcDNA3.1/HisB (Invitrogen Corporation, Carlsbad, CA), respectively. All constructs were prepared by standard alkaline lysis and two rounds of caesium chloride gradient purification.

Immunofluorescence analysis

COS7 cells were transiently co-transfected with pFLAG-GRIP1{tau} and pcDNA3.1 HisB/SII-T1 using Lipofectamine 2000 (Invitrogen), and processed as previously described (Saso et al. 2003). Fluorescence images were obtained with an Olympus BH2-RFCA microscope. Multiple images were collected and representative images are shown. All images were digitally processed for presentation using Adobe Photoshop.

Co-immunoprecipitation assays

COS7 cells were maintained as previously described (Saso et al. 2003). Approximately 1.6 x 106 cells were co-transfected with 2 µg of FLAG-tagged GRIP1{tau} expression vector and 6.8 µg of Xpress-tagged SII-T1 expression vector using Lipofectamine 2000. After incubation for 48 h, the cells were harvested, lysed for 20 min in lysis buffer [50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, Complete Mini protease inhibitor tablet (Roche Diagnostics, Mannheim, Germany)]. After centrifugation at 15 000 g for 30 min at 4 °C, anti-FLAG M2 Affinity Gel (Sigma-Aldrich Chemical Co., St Louis, MO) was added to the cleared lysate and incubated at 4 °C overnight. The immunoprecipitates were washed twice with the wash buffer (50 mM Tris/HCl, pH 7.4, 450 mM NaCl, 0.5% Triton X-100, 1 mM EDTA) and subjected to immunoblotting with anti-Xpress antibody (Invitrogen) or anti-FLAG antibody (Sigma-Aldrich).

Luciferase reporter assays

STO embryonic fibroblast cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% foetal calf serum, 50 U/mL penicillin, 50 µg/mL streptomycin (Invitrogen). Transfections were performed using Lipofectamine 2000 with 950 ng of the pG5luc reporter plasmid (Promega), 50 ng of effector construct pBIND (encoding GAL4DBD alone) or GAL4DBD-GRIP1{tau} derivatives, and FLAG-tagged SII-T1 expression vector where indicated. Cells were lysed in Passive Lysis Buffer (Promega) 48 h after transfection and firefly and renilla luciferase activity were assayed using the Dual Luciferase Assay System (Promega). All firefly luciferase activity values were normalized to renilla luciferase activity values. All transfections were performed in duplicate.


   Acknowledgements
 
This work was supported by grants-in-aid for scientific research from the Japan Society for the Promotion of Science.


   Footnotes
 
Communicated by: Hiroshi Handa

* Correspondence: E-mail: sekimizu{at}mol.f.u-tokyo.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 10 July 2004
Accepted: 24 August 2004




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