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1 Department of Molecular Genetics, and 2 Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita City, Osaka, Japan
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and Tisp40ß, both of which contain a transmembrane domain and localize to the endoplasmic reticulum (ER). In contrast, mutant proteins lacking the transmembrane domain (Tisp40/ß
TM) primarily localize to the nucleus. Endoglycosidase H treatment shows that the C-terminus of Tisp40/ß is glycosylated. Protease experiments demonstrate that Tisp40/ß are Type II transmembrane proteins that are released into the nucleus by a two-step cleavage mechanism called ‘regulated intramembrane proteolysis’ (Rip). Unlike previously published observations, Tisp40 does not bind to the NF-
B site; instead, it specifically binds to the unfolded protein response element (UPRE). Luciferase assays reveal that Tisp40ßTM activates transcription through UPRE. Northern blot analysis shows that Tisp40/ßTM proteins up-regulate EDEM (ER degradation of enhancing -manosidase-like protein) mRNA. These observations unveil a novel event in mouse spermiogenesis and show that the final stage of trans-criptional regulation is controlled by the Rip pathway. |
Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialReferences |
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Two bZip-type transcription factors, CREB and CREM, are reported to play pivotal roles in the events that occur before these morphological changes in spermatogenesis (Sassone-Corsi 2000). The CREB family genes in general are known to undergo alternative splicing that generates many kinds of isoforms, with a notable example in the male germ cell, which expresses dominant-negative forms of both CREB (
and , lacking the bZip domain) and CREM (, ß, and , lacking the activation domain) in the early meiotic stages. Expression of CREM
(the active form) is very low in infant testis, whereas CREM expression is very high in adult testis. The active transcription factor CREM, which is mainly expressed in the round spermatids and secondary spermatocytes, up-regulates the transcription of at least four spermiogenic genes, namely, RT7, Tnp1, ACE, and calspermin (Nantel et al. 1996). In the CREM (–/–) testis, apoptotic germ cells are significantly increased and no late spermatids are found. Thus, CREM is essential for spermatogenesis (Blendy et al. 1996; Nantel et al. 1996).
The Rip (regulated intramembrane proteolysis) pathway is a conserved mechanism of protein activation found in organisms ranging from bacteria to humans; it plays a pivotal role in sterol metabolism and UPR (Unfolded Protein Response) (Ye et al. 2000). In UPR, ATF6 is activated (cleaved) by a specific Rip pathway, and activates the expressions of endoplasmic reticulum (ER) chaperones that support protein folding and XBP-1 through the ER stress response element (ERSE) (Mori 2003). While unspliced XBP-1 cannot act as an active transcription factor, the spliced form becomes highly active and up-regulates EDEM (ER degradation of enhancing -manosidase-like protein), which enhances unfolded protein degradation, presumably through UPRE (unfolded protein response element) (Oda et al. 2003; Yoshida et al. 2003).
We previously isolated 80 kinds of TISP (transcript induced in spermiogenesis) genes whose transcripts are dramatically induced in spermiogenesis by the step-wise subtraction technique. That there were so many was a surprise because it had been believed that almost no nascent transcription occurs after meiosis (Fujii et al. 2002). Many of the TISP genes were novel and functional analyses on these genes are currently expanding our understanding of spermiogenesis (Fujii et al. 1999; Tosaka et al. 2000). We found that one of these TISP genes, TISP40, encodes a bZip-type membrane-bound transcription factor whose expression is restricted to the haploid spermatids after CREM expression. Here we report that TISP40 is expressed during spermiogenesis and that Tisp40 functions through UPRE and is activated via the Rip pathway.
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Results |
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and Tisp40ß are exclusively expressed in the testis
We previously reported TISP40 is one of the novel genes whose transcription is dramatically induced during mouse spermiogenesis (Fujii et al. 2002). Later, another group showed that Tisp40/Atce1 is exclusively expressed in postmeiotic cells as a 1.3 kilobase (kb) transcript (Stelzer & Don 2002). Since then, a new TISP40 cDNA subtype harboring an extra 5' portion was reported in the NCBI data bank (BC022605
[GenBank]
). We obtained this cDNA subtype, which encodes a 370 amino acid protein by RT-PCR; it contains an extra 5' region that adds 55 acidic amino acids to the N-terminus of the original Tisp40 protein. Thus, we hereafter denote the original Tisp40 protein as Tisp40 and the new subtype as Tisp40ß.
Tisp40 (315 amino acids) harbors a domain rich in basic amino acids followed by a leucine zipper motif, whose structure is commonly found among bZip transcription factors. A dendogram analysis was performed using the amino acid sequences of Tisp40ß relatives such as Mus musculus (Mm) Tisp40ß (BC022605
[GenBank]
), Homo sapiens (Hs) AIbZIP (BC038962
[GenBank]
), HsCREB-H (2716279), MmCREB3 (BC002094
[GenBank]
), MmOASIS 2519368, MmCREB (M95106
[GenBank]
), MmCREM (M60285
[GenBank]
), MmATF6 (AK36335) and MmATF6ß (AB10266). The result revealed that MmTisp40, HsAIbZIP, HsCREB-H and MmCREB3 separate into a distinct subgroup of ATF/CREB/CREM proteins (Fig. 1A). MmTisp40 is most akin (66% identity) to HsAIbZIP (Qi et al. 2002). Thus, Tisp40 is a bZip-type transcription factor that is akin to HsAIbZip, HsCREB-H and MmCREB3.
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and Tisp40ß, probably because their size difference (165 nucleotides) is within the range of the randomly sized poly(A) tail of the mRNA molecules (about 300 nucleotides). We also analyzed the transcription of mouse TISP40 with RNA from testes that cannot produce differentiated germ cells, namely, those in cryptorchid, jsd/jsd, Sl17H/Sl17H and W/WV mice (Fujii et al. 2002). As expected, TISP40 is expressed at almost undetectable levels in these testes, further emphasizing the specific role TISP40 plays in mouse spermiogenesis. Northern blots using RNAs from mice of various ages indicated that TISP40 gene expression was very low in the prepubertal testis (2–5-, 8- and 17-day-old) but became abruptly induced in the postpubertal testis (29-day-old), after which expression increased (35- and 60-day-old testes), as shown in Fig. 1B,ii. This suggests that TISP40 transcription is induced after meiosis. Northern analysis using total RNA purified from fractionated mouse testicular cells (the germ, Leydig and Sertoli cells) showed that TISP40 mRNA was exclusively expressed in the germ cells (Fig. 1B,iii).
To analyze the potentially different expression of the TISP40 and TISP40ß mRNAs, we performed RT-PCR analysis using a pair of primers that can distinguish between them. As shown in Fig. 1C, the bands for the TISP40 and TISP40ß mRNAs were exclusively detected in the testis. Notably, the TISP40ß band was barely detectable after the first round of RT-PCR employing 30 amplifications (Fig. 1C, second panel). Thus, we recovered the amplified DNA fragment from the reaction tube by phenol extraction followed by ethanol precipitation and used it as the substrate for a second round of RT-PCR employing 30 amplifications. This indicated that the TISP40ß mRNA band was only detected in the testis, like TISP40 mRNA. Thus, it appears that TISP40ß mRNA is expressed at very low levels in the testis. No band was detected in the mouse embryo of age 7–17-days.
To determine the developmental stages of the germ cells in the adult testis that specifically express TISP40 mRNA, we performed in situ hybridization (ISH) analysis on an adult testis using anti-sense and sense cRNA probes generated from TISP40 cDNA. As shown in Fig. 1D,E, we observed specific staining with the anti-sense probe alone, primarily in round spermatid layer and not in the elongated spermatid layer of the seminiferous tubules from stage V to stage VIII. Other germ cells and supportive cells showed no positive staining. Thus, the TISP40 transcripts exist primarily in the step 5 to step 8 spermatids. Step 5 spermatids are present in at least the 22-day-old testis; however, as shown by Fig. 1B,ii, we could not detect Tisp40 mRNA in the 23-day-old testis, although it was detected in the 29-day-old testis (Fig. 1D and Kluin et al. 1982). This discrepancy between the ISH and Northern blot data is probably because the expression of Tisp40 protein is only gradually increased after the second wave of spermatogenesis; as a result, TISP40 mRNA expression levels are very low in 23-day-old mice and could not be detected by Northern analysis. Supporting this is Western blot analysis that showed Tisp40 protein levels are only up-regulated in 6-month-old mice and are not observed in 1- or 2- month-old mice (data not shown). Thus, we speculate that TISP40 mRNA is translated as early as after 22 days.
Tisp40/ß proteins are specifically expressed in the postmeiotic testis
To characterize Tisp40 protein expression, we raised several polyclonal rabbit antibodies against Tisp40/ß using the N-terminal portion of Tisp40 (amino acids #1–130) (shown as ‘epitope’ in Fig. 2A). We then first expressed Myc-tagged Tisp40 or Tisp40ß or their truncated forms lacking the transmembrane domain (TM) (depicted schematically in Fig. 2A) in HeLa cells and confirmed their expression by Western blot analysis using an anti-Myc antibody (Fig. 2B). When we probed the duplicated Western blots with the anti-Tisp40/ß antibody we raised in three different rabbits, we found that one, TI-3, specifically recognized Tisp40/ß (Fig. 2C). Thus, we used TI-3 in the following experiments.
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and Tisp40ß consist of 315 and 370 amino acids with calculated molecular weights of 35 and 41 kDa, respectively, these proteins migrated slightly slower than expected. This is probably due to glycosylation as will be shown in Fig. 4 below. The proteins are not present in any of the somatic tissues, although a nonspecific band is recognized in the liver by the TI-3 antibody. With regard to Tisp40 and Tisp40ß protein expression during development, both were expressed at very low levels in the prepubertal testis (5-, 8-, and 17-day-old testes) but are detected strongly in the postpubertal testis (29-, 35-, and 60-day-old testes). A nonspecific band for Tisp40ß is observed in the 5- and 8-day-old testes. Notably, Tisp40 is expressed after Tisp40ß (Fig. 2E). Moreover, the intensity of the band for Tisp40ß is stronger than that of Tisp40, which contrasts with the much smaller amount of TISP40ß mRNA observed in the testis compared to the TISP40 mRNA levels shown in Fig. 1C. This result suggests that TISP40ß mRNA is translated much more efficiently than TISP40 mRNA.
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/ß are localized to the perinuclearplasm but not in the nucleus of HeLa cells
Since we could not observe Tisp40/ß in the testis by immunofluorescence microscopy using the TI-3 antibody, we investigated the subcellular localization of Tisp40/ß in HeLa S3 cells. We thus transiently transfected HeLa cells with the plasmids that express fusion proteins of 6Myc at the N-termini of the full-length Tisp40/ß proteins and the truncated Tisp40/ßTM proteins. Indirect immunofluorescence analysis with counterstaining with 4',6-diamidino-2-phenylindole (DAPI) showed that both the truncated Tisp40TM and Tisp40ßTM proteins entered the nucleus (Fig. 3A). In contrast, intact Tisp40 and Tisp40ß were detected in the perinuclearplasm, which consists of the ER, Golgi and cytoplasm. We confirmed that the mutant forms of Tisp40/ß were actually expressed in transfected HeLa cells by examining the sizes of their expressed proteins by Western blot analysis (Fig. 3B). Thus, Tisp40 and Tisp40ß are usually retained in the perinuclearplasm but deletion of the TM domain causes them to move into the nucleus due to the nuclear localization signal (NLS) found in the middle of their molecules (Fig. 2A).
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tagged with three motifs (- Flag, -His6 and -HA) in tandem at its C-terminus and performed double-labeling immunofluorescence on HeLa cells transfected with this plasmid using the ER-staining anti-KDEL antibody (Haze et al. 1999) and anti-HA antibody. The localizations of the two antibodies were visualized by Alexa488 (green)- and Alexa 594 (red)-conjugated secondary antibodies, respectively. The overlay of the green (KDEL) and red (Tisp40) images revealed the almost complete co-localization of the ER and Tips40 signals (Fig. 3C). Thus, Tisp40 is localized to the ER. We also confirmed the co-localization of the N-terminal 6Myc and C-terminal HA staining signals (data not shown).
Post-translational modifications of Tisp40/ß
To determine the orientation of the Tisp40/ß proteins in the perinuclearplasm, we expressed the 6Myc-Tisp40-Flag-His6-HA protein in HeLa cells, obtained the cell extract, and removed the nuclear fraction by centrifugation. The supernatant containing the membrane fraction was incubated with or without various amounts of trypsin, followed by Western blot analysis with the PL14 anti-Myc or the 3F10 anti-HA antibody to detect the Tisp40 N-terminus or C-terminus, respectively. At the concentration of trypsin that digested the full-length Tisp40 protein almost completely, only the anti-HA antibody detected a smaller band, which corresponds to Tisp40 containing an intact C-terminus (Fig. 4A; middle panel, lane 4). In contrast, the anti-Myc antibody did not detect a band at all at this trypsin concentration (Fig. 4A; lane 4, uppermost panel). We confirmed that this trypsin sensitivity of the C-terminus but not the N-terminus was increased by treatment with 1% Triton x100 (lanes 5–8 in uppermost and middle panels). We also examined the trypsin sensitivity of the N- and C-termini of calnexin as a control. Calnexin is a Type I transmembrane protein whose N-terminus resides in the ER lumen. The protected N-terminus of calnexin was detected in the absence of 1% Triton X100 (Fig. 4A, third panel from the top, lanes 3 and 4), but no protected C-terminus band was detected in the presence or absence of Triton x100 (Fig. 4A, lowermost panel, lanes 3, 4, 7 and 8). Thus, the N-terminus of Tisp40, which contains the bZip domain, is in the cytoplasm, whereas the C-terminus is in the ER lumen, where it is protected from digestion by trypsin.
To determine if Tisp40/ß are glycosylated in HeLa cells, we conducted a glycosidase sensitivity assay. In the absence of Endoglycosidase H, 6Myc-Tisp40 expressed in HeLa cells migrated as 58 and 64 kDa bands (Fig. 4B, lane 2), while 6Myc-Tisp40ß migrated as 70 and 76 kDa bands (Fig. 4B, lane 5). However, the more slowly migrating bands disappeared after treatment with Endoglycosidase H (Fig. 4B, lanes 3 and 6, respectively). Thus, we conclude that Tisp40/ß are glycosylated in HeLa cells somewhere in their C-terminal portions, possibly at a putative glycosylation motif. Candidate sites for N-glycosylation in Tisp40 are N-glycosylated motifs (Asn-X-Ser/Thr) found at amino acids 263 and 287. Notably, when we examined the testis extracts (Fig. 4C; lane 4) for these slowly migrating Tisp40 and Tisp40ß bands, we could not detect them. In this experiment, in vitro translated untagged Tisp40 and Tisp40ß proteins were also electrophoresed as a control (Fig. 4C; lanes 2 and 3). This suggests that the level of N-glycosylation of these proteins in the testis is low.
Tisp40 is processed via the Rip pathway
It is known that transcription factors such as activating transcription factor 6 (ATF6) and sterol response element-binding protein (SREBP) contain a transmembrane domain that allows them to localize to the ER (Rawson et al. 1997; Haze et al. 1999; Ye et al. 2000). Upon ER stress or sterol deficiency, these transcription factors are translocated to the Golgi apparatus, where they are sequentially cleaved by Site 1 protease (S1P) and Site 2 protease (S2P), and then released from the membrane into the nucleus to induce the transcription of their targets genes (Brown et al. 2000; Ye et al. 2000). This regulatory mechanism is called Rip, and it is conserved in organisms ranging from bacteria to humans (Brown et al. 2000).
Since Tisp40/ß localize to the ER rather than in the nucleus (Fig. 3A,C), it will be necessary that they trans-localize into the nucleus to regulate their target gene expression. When we expressed Tisp40/ß using a strong CMV promoter, we found that the Tisp40/ß proteins are cleaved even without ER stress signals, as shown by the arrows or arrowhead in lanes 5 and 9 of Fig. 3B. The upper band shown by an arrowhead and arrow in lanes 5 and 9, respectively, may be due to S1P cleavage, while the lower band in lane 5 shown by an arrow may be derived from an additional cleavage by S2P. To overcome the background cleavage of the proteins when expressed from the CMV promoter, we inserted TISP40/ß cDNA into the pEGFP3B(s) vector, which is identical to pCMVshort-EGFP except that the multicloning site is taken from the pEGFP3B vector (Fujii et al. 1999). The advantage of using this truncated CMV promoter is that it allows us to attain lower expression level of the fused gene. Tisp40/ß expressed from this vector are not cleaved without a signal (Fig. 5A, lanes 2, 3, 5 and 6). Using this construct, we then transfected 293t cells with pEGFP3B(s)-Tisp40 and harvested the cell lysates 48 h later, followed by Western blot analysis. Tisp40 was not cleaved unless the pCMV-S1PTM-KDEL construct was co-expressed, which expresses a functional S1P protein tagged with the KDEL sequence that facilitates its localization into the ER (Fig. 5B, lanes 2 and 3) (DeBose-Boyd et al. 1999). We then assessed the effect of co-transfecting mutant forms of S1P, namely, those that bear the KDAS sequence instead of KDEL and/or have an Ala substitution in the Ser-414 residue. Replacement of KDEL with KDAS generates a non-functional mutant. The S414A point mutation also produces an inactive form of S1P (Sakai et al. 1998). None of the mutant forms of S1P induced Tisp40 cleavage (Fig. 5B, lanes 4–6). We have also detected Tisp40 in the nucleus of the adult testis (the final destination for proteins regulated by Rip) but not in the cytoplasm (data not shown). Taken together, these data indicate that Tisp40/ß proteins are cleaved by at least S1P and that a Rip pathway participates at the mid-stage of spermiogenesis.
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/ß-transfected 293t cells has on Tisp40 cleavage, since BFA promotes the trans-localization of active endogenous S1P and S2P from the Golgi apparatus to the ER (Raggo et al. 2002), which is where Tisp40/ß is localized. Western blot analysis of cell lysates harvested 48 h after transfection with or without BFA with anti-GFP antibody showed that BFA enhanced the cleavage of GFP-Tisp40 (Fig. 5D, lanes 2 and 3) and that the migration of this cleaved GFP-Tisp40 product is similar to that of GFP-Tisp40TM (Fig. 5D, lane 1). However, since the cleavage efficiency of GFP-Tisp40 in the presence of BFA was very low, it was difficult to analyze the Rip mechanism in more detail. To overcome this problem, we prepared five C-terminal deletion mutants of Tisp40 (Tisp40/ßC6-C10). One of these, Tisp40C8, which is depicted in Fig. 5C, displayed a high rate of cleavage with or without BFA (Fig. 5D, lanes 4 and 5, and data not shown).
Next, we compared the amino acid sequences of human SREBP-2, human ATF6 and mouse Tisp40, and found that Tisp40 contains the S1P and S2P recognition motifs RXXL and LXXXXLXXXP, respectively (Raggo et al. 2002) (Fig. 5E). Thus, we generated point mutant forms of Tisp40/ßC8 that had substitutions in either of these motifs. This yielded the Arg256Ala and Leu232Pro mutants of Tisp40C8 (substituted in the S1P and S2P motifs, respectively), and the Arg311Ala and Leu287Pro mutants of Tisp40ßC8 (substituted in the S1P and S2P motifs, respectively). 293t cells were transfected with one of these mutants and subjected to Western blot analysis with anti-GFP antibody. We found that while the GFP-Tisp40/ßC8 proteins were cleaved, the proteins with a mutation in their S1P motif were not (Fig. 5F, lanes 3, 4, 8, and 9). The proteins bearing the mutation in the S2P motif continued to be cleaved but the migration of the cleaved product became slightly slower compared to the cleaved GFP-Tisp40/ßC8 product (Fig. 5F, lanes 5 and 10). This is probably because these mutant proteins were cleaved by S1P but not S2P. These data indicate that Tisp40 is processed in the Rip pathway at the mid-stage of spermiogenesis and that S1P is more involved in this process than S2P.
Tisp40 binds to UPRE but not to CRE or the NF-B site
It is known that many CREB family proteins bind to the consensus CRE sequence. In contrast, Stelzer & Don (2002) reported that Atce1 (Tisp40) bound to the NF-B site of the IL2 receptor gene, whose core sequence is quite distinct from the CRE sequence. To determine the element bound by Tisp40, we performed an electrophoresis mobility shift assay (EMSA) using the nuclear form of Tisp40 (Tisp40TM) and oligonucleotides bearing various binding elements. Unlike the previous report, we found that Tisp40TM did not bind to either CRE of the somatostatin gene or the NF-B site of the IL2 receptor gene (Fig. 6A, lanes 4 and 6). Instead, it preferentially bound to UPRE (Fig. 6A, lane 2). We also confirmed that the oligonucleotides bearing the NF-B or CRE sites were functional since nuclear extracts prepared from TNF--treated HeLa S3 cells bound to the NF-B site (Fig. 6B, lane 3) and in vitro-expressed HA-tagged CREM protein bound to the CRE site (Fig. 6C, lane 2). A super-shift assay with anti-HA antibody (3F10) confirmed that the protein complex bound to the UPRE probe actually contained Tisp40TM (Fig. 6D, lane 3). Successful competition using a 50- or 500-fold molar excess of unlabeled UPRE probe also confirmed the specific association of Tisp40TM with UPRE (Fig. 6E, lanes 3 and 4). In contrast, the unlabeled CRE or NF-B site probes did not show such efficient inhibition of binding (Fig. 6E, lanes 5–8). Competition assays to compare the binding ability of Tisp40TM to wild-type (wt) UPRE (TGACGTGG) and mutant (mt) UPRE (TGACGTTG) also showed that while wt UPRE efficiently reduced the binding activity of Tisp40TM to UPRE, the mutant was much less efficient (Fig. 6F, lanes 3–8).
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TM by using competitor UPRE oligonucleotides that harbor point mutations. As shown in Fig. 6G, a nucleotide change from G to T at nucleotide numbers 17 (lane 6) or 23 (lane 12) most effectively inhibited the binding of Tisp40TM to UPRE. A change from G to A at nucleotide number 22 also induced very effective competition (Fig. 6H, lane 9). Taken together, we conclude that, of the sequences examined to date, Tisp40TM preferentially binds to the T(G/T)ACGT(G/A)(G/T) sequence. The Tisp40ß-specific acidic domain is required for transcriptional activity
To investigate whether Tisp40/ß proteins are transcriptional activators, we conducted luciferase assays. Thus, expression plasmids were constructed on the basis of pBIND, which carries the GAL4 DNA-binding domain and expresses Renilla luciferase driven by the SV40 promoter. Various regions of Tisp40/ß were amplified by PCR and inserted into the AscI/NotI sites of pBIND (depicted in Fig. 7A) and co-transfected with a pG5 reporter plasmid into HeLa cells. Transcriptional activity was measured by using the dual luciferase reporter system (Promega). All the GAL4-Tisp40 constructs containing the N-terminal acidic domain that is specific to Tisp40ß could activate reporter expression (Fig. 7A): these included the intact Tisp40ß construct and the C-terminally truncated Tisp40ßSD (ß-specific domain), Tisp40ßbZip, and Tisp40ßTM constructs. In contrast, the GAL4-Tisp40 constructs lacking the N-terminal acidic domain could not activate reporter expression (Fig. 7A): these included the intact Tisp40 construct and the Tisp40N, Tisp40NN, Tisp40NNN, Tisp40bZip, and Tisp40TM constructs. Thus, we conclude that Tisp40 is inactive and Tisp40ß alone functions as an active transcription factor. We also confirmed the expression of each of the Tisp40 constructs by Western blot analysis (Fig. 7B).
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TM activates transcription through UPRE
To investigate whether Tisp40/ß activates transcription through UPRE or ERSE, we co-transfected Tisp40/ß-expressing vectors separately with either the pGL3-GRP78(- 132)-luc or p5XUPRE-GL3-luc reporter vector. As shown in Fig. 7C, Tisp40ß but not Tisp40 could activate reporter expression through UPRE by 5-fold relative to the control (Fig. 7C, lanes 3 and 5). Furthermore, the nuclear form of Tisp40ß (Tisp40ßTM) could activate reporter expression by eight-fold through UPRE compared to the control (Fig. 7C, lane 4), even though its expression level is much lower than the others, as shown by Western blot analysis (Fig. 7E, lane 4). In contrast, Tisp40ß or its TM form showed equivalently low transcriptional activation to Tisp40 or its TM form through ERSE, indicating that Tisp40/ß cannot activate transcription through ERSE (Fig. 7D). This contrasts with a previous report that showed overproduced ATF6 and XBP1 activate transcription not only through UPRE but also through ERSE (Wang et al. 2000; Yoshida et al. 2001).
Yoshida et al. (2003) demonstrated that XBP1 enhances the transcription of the EDEM gene, and that this was presumably mediated by UPRE. We therefore surmised that Tisp40 and/or Tisp40ß may regulate the transcription of EDEM through putative UPRE. To test this, we transiently transfected HeLa cells with a plasmid expressing the indicated Tisp40/ß construct and investigated the expression of EDEM by Northern blot analysis (Fig. 7F). We found that EDEM mRNA expression was strongly induced by overproducing Tisp40ßTM. We also found that Tisp40TM could slightly induce the expression of EDEM mRNA. Finally, we confirmed that the two Tisp40 mutants cannot up-regulate Grp78/BiP, an ERSE-regulated gene (Fig. 7G).
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSupplementary materialReferences |
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During spermiogenesis, germ cells are subjected to dramatic morphological changes that facilitate their acquisition of sperm fertility and mobility. These changes involve chromatin condensation that is directed by the replacement of histones with transition proteins or protamines (Hecht 1998; Eddy 1999). This chromatin condensation causes the complete shut down of new transcription, and CREM alone is thought to be the key transcriptional regulator during the process of spermatogenesis (Sassone-Corsi 2002a). In the present study, however, we show that the TISP40 gene is exclusively expressed at a later stage than the one during which CREM functions (Fig. 1). The TISP40 gene generates two kinds of proteins named Tisp40 and Tisp40ß, both of which are expressed exclusively in haploid germ cells (Fig. 2). Tisp40/Atce1, which was previously isolated by screening a mouse testis library with a two-hybrid approach (Stelzer & Don 2002), corresponds to Tisp40. Our protease protection assay (Fig. 4A), Endoglycosidase H assay (Fig. 4B), and immunofluorescence analysis (Fig. 3A,C) revealed that the Tisp40/ß proteins are glycosylated Type II transmembrane proteins that are embedded in the ER membrane in HeLa cells.
Tisp40 is regulated via the Rip pathway
As described above, it is believed that transcription in late spermatids is silenced due to chromatin condensation. This notion is challenged by our discovery of Tisp40, a bZip-type transcription factor whose expression starts specifically in the mid spermatid stage. It is curious that Tisp40/ß proteins are normally localized to the ER membrane and do not localize to the nucleus, which suggests that if these proteins act as transcription factors, they will have to be truncated into a form lacking the transmembrane domain (Tisp40TM) that anchors them to the ER; this would allow them to enter the nucleus. We propose here that Tisp40/ß proteins are originally expressed as inactive form embedded in the ER membrane. When a certain stress response signal is emitted at the mid-stage of spermiogenesis, the Tisp40/ß proteins are released from the ER membrane by cleavage with S1P and S2P via a two-step cleavage mechanism called Rip (Rawson 2002). This allows them to enter the nucleus, whereupon they can regulate the transcription of their target gene(s). Supporting this hypothesis is that Tisp40 is present in the adult testis in a nuclear form whose size is similar to the untagged Tisp40/ßTM protein observed by Western blot analysis (data not shown).
Similar regulatory mechanisms to the one proposed above for Tisp40 have been reported for ATF6, SREBPs and Luman (Rawson et al. 1997; Brown et al. 2000; Ye et al. 2000; Raggo et al. 2002). ATF6 and SREBPs are activated by proteolytic cleavage, and the ensuing nuclear translocation of their N-termini activates their respective target genes, which are involved in UPR and lipogenesis (Haze et al. 1999; Zeng et al. 2004). Similarly, Tisp40/ß may be translocated into the nucleus upon their cleavage because they harbor NLS in the middle of the molecule. Indeed, disruption of the NLS abolishes their nuclear localization in HeLa cells (Fig. S1). New membrane-bound transcription factors have been recently reported: these include CREB-H, AIbZip, and Oasis (Omori et al. 2001, 2002; Qi et al. 2002). However, it remains unclear whether they are also cleaved by S1P.
Tisp40/ß proteins regulate transcription through UPRE
Many male haploid germ cell-specific genes contain the CRE sequence in their promoter region. However, we found Tisp40/ß interacted only weakly with the CRE site of somatostatin (Fig. 6). Stelzer & Don (2002) have reported that Atce1 (Tisp40) binds to the NF-B site of the IL2 receptor gene, which is peculiar considering its CREM-like structure (Sassone-Corsi 2002b). In the present study, we demonstrate that Tisp40 does not bind to the NF-B site. In contrast, it preferentially binds to UPRE (Fig. 6A), and the protein/UPRE site complex contains Tisp40/ß, as shown by a super-shift assay (Fig. 6D). Competition EMSA analyses also revealed that the UPRE-like site that Tisp40/ß binds to with the greatest affinity is T(G/T)ACGT(G/A)(G/T) (Fig. 6F–H).
ATF6 and XBP1 also bind to ERSE (CCAATN9 CCACG) through cooperation with the NF-Y trimer. We surmised that Tisp40/ß may thus also regulate the expression of the GRP78/BiP, GRP94, and calreticulin genes, as they carry ERSE in their promoter regions. Our experiments revealed, however, that Tisp40/ß does not activate gene transcription through ERSE (Fig. 7D). Supporting this is that GRP78/BiP mRNA expression was not altered when Tisp40/ß were over-expressed (Fig. 7G). Thus, we conclude that the Tisp40/ß proteins regulate transcription through UPRE but not through ERSE.
We also found here that Tisp40ßTM expression induced EDEM mRNA expression (Fig. 7F). Since we could not find UPRE in the promoter region of EDEM, we speculate that UPRE may serve as an enhancer of EDEM expression. This notion is supported by a recent report showing that the induced expression of the UPR gene C/EBPß (CCAAT/enhancer-binding protein ß) is mediated by XBP1 through an UPRE-like element found in the 3' untranslated region (3'UTR) of C/EBPß (Chen et al. 2004). It remains unclear whether the Tisp40ß-induced transcription of EDEM is due to direct regulation through UPRE or merely an indirect phenomenon.
Why the Rip pathway at the mid-stage of spermiogenesis?
We show here that Tisp40ßTM but not Tisp40TM is transcriptionally active. Nonetheless, Tisp40TM induced the expression of EDEM (Fig. 7F). Our preliminary data suggest that the Tisp40TM homodimer does not bear a transcriptional activation domain, whereas the formation of a heterodimer between Tisp40TM and CREM can activate transcription through CRE, at which point both proteins lose their ability to bind to UPRE (unpublished observation). It is feasible that a CRE-like element is required for the transcriptional induction of EDEM. Supporting this is one of the UPR genes, ATF4, which encodes a candidate protein that induces GRP78/Bip after ER stress: this protein is activated by UPR and binds to the CRE motif (Luo et al. 2003).
UPR is widely conserved from bacteria to mammals and abnormal handling of UPR cause Alzheimer's and Parkinson disease in humans (Urano et al. 2000; Patil & Walter 2001; Kaufman 2002). UPR is regulated in budding yeast by the IRE1-HAC1 pathway, whereas three pathways regulate it in mammals, namely, the IRE1-XBP1 (mammalian HAC1), ATF6, and PERK (PKR ER kinase) pathways (Mori 2003). These pathways play distinct roles in UPR and it has been proposed that they participate separately in a time-dependent phase shift, where an ATF6-mediated refolding only phase is followed by an XBP1-mediated refolding plus degradation phase (Mori 2003; Yoshida et al. 2003).
Since a large amount of proteins are produced during spermiogenesis to constantly generate sperm, the UPR activation mechanism plays a pivotal role in monitoring and removing abnormally unfolded proteins. Supporting this is that the expression of some chaperones are induced during spermiogenesis and disruption in these genes cause male infertility due to cell cycle arrest and apoptosis (Eddy 1999). Similarly, the disruption of calmegin, a testis-specific lectin chaperone, also induces male infertility (Ikawa et al. 1997). The accumulation of unfolded proteins due to impaired removal by UPR causes apoptosis in somatic cells, which is mediated by an ER stress-specific caspase, caspase-12 (Nakagawa et al. 2000). The apoptosis of a single spermatid is dangerous for the surrounding spermatids because their cytoplasms are linked by a cytosolic bridge, and thus the caspase emitted from a single cell may rapidly circulate through all the spermatids. Thus, we suppose that spermatids need to accumulate large amount of chaperones before they respond to urgent ER stress that may occur during spermiogenesis. We propose that the membrane-bound Tisp40/ß proteins are likely to be on constant alert for ER stress that will cause them to induce the UPR genes, including EDEM, that then induce the expression of spermatid-specific chaperones. This assumption will be tested in the near future.
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To prepare pCMV-6Myc-Tisp40/ß, -Tisp40/ßTM and plasmids expressing Tisp40/ß deletion mutants, we generated DNA fragments for the relevant open reading frames (ORFs) by PCR. The oligonucleotides used in these experiments are listed in Table 1. The absence of PCR-induced point mutations was confirmed by DNA sequencing. pEGFP3Bshort is identical to pCMVshort-EGFP-C1 (Nadanaka et al. 2004) except for its multicloning site, which is derived from the BglII-KpnI fragment pEGFP3B. p5XATF6 (UPRE)-GL3 and pGL3-GRP78P (- 132)-luc are kind gifts from Prof K. Mori (Kyoto University).
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Preparation of RNA from various tissues and the fractionation of germ, Leydig, and Sertoli cells from adult mice and Northern blot analysis were performed as previously described (Fujii et al. 1999). The cDNA inserts cut out of the plasmids by the SmaI/NotI restriction enzymes were radiolabeled with 32P-dCTP using the Random Primer DNA Labeling kit (TaKaRa, Japan) and used as hybridization probes. Total RNA (5 µg/µL) was extracted from various tissues and from the testes of C57BL/6 mice at various ages. A Northern blot with radiolabelled GAPDH cDNA served as a loading control. We used Mm Tisp40-specific 5'UTR and 3'Tisp40TMNotI to detect Tisp40 and Mm Tisp40ß-specific 5'UTR and Mm 3'Tisp40 RT to detect Tisp40ß. We employed a multitissue cDNA panel as a template for the RT-PCR analysis (Clontech, Palo Alto, CA, USA). Procedures for in situ hybridization have been previously described (Fujii et al. 1999).
Western blot analysis
Extracts of various C57BL/6 mouse organs, HeLa S3, and 293t cells lyzed in RIPA buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, 0.1% sodium deoxycholate, and 1 mM PMSF) were centrifuged and the supernatant was quantified by the Bradford method (Nacalai, Kyoto, Japan). About 50 µg of protein (tissue extracts) per lane were separated by 12% SDS-PAGE and transferred to polyvinylidine difluoride (PVDF) filters (Millipore, Bedford, MA, USA). The filters were blocked overnight at 4 °C with TBS-T containing 10% (or 5%) skim milk and then incubated at room temperature for 4 h with primary antibody diluted at 1 : 1000 (for anti-Myc antibody (PL14) and anti-HA antibody (3F10)) or at 1 : 10 000 (for anti-Tisp40 antibody (TI-3)) in TBS-T containing 5% or 10% skim milk. The filters were then washed three times in TBS-T for 7 min, incubated at room temperature for 2 h with a horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG (Amersham Pharmacia Biotech, Piscataway, NJ, USA) or goat anti-mouse IgG or rat IgG (Cappel, West Chester, PA, USA)) diluted 1 : 1000 in TBS-T containing 5% skim milk, and washed three times in TBS-T for 10 min. A chemiluminescent blotting substrate (PerkinElmer Life Science, Boston, MA, USA) was used for the detection step according to the manufacturer's recommendations.
Electrophoresis mobility shift assay
6Myc-Tisp40TM, HA-Tisp40TM, and HA-CREM were expressed in vitro using the TNT system (Promega). HeLa S3 cells treated with or without TNF- for 30 min were washed with 3 mL PBS(-), centrifuged 1000 g for 5 min at 4 °C and the supernatant was removed. The cell pellets were suspended in 2 mL NP40 lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.5% NP40) containing a protease inhibitor cocktail (Sigma) for 10 min on ice. These cell lysates were then centrifuged at 17 400 g for 5 min at 4 °C and the supernatant was removed. The pellets (nuclear fraction) were washed twice with NP40 lysis buffer. The cell pellets were resuspended in 100 µL glycerol storage buffer (50 mM Tris-HCl pH 8.0, 40% Glycerol, 5 mM MgCl2, 0.1 mM EDTA) containing a protease inhibitor cocktail (Sigma) and stored at –80 °C. Complementary (forward and reverse) oligonucleotides corresponding to specific binding elements were synthesized and labeled with [-32P]ATP by using T4 polynucleotide kinase (TOYOBO, Osaka, Japan) in T4 polynucleotide kinase buffer [50 mM Tris-HCl pH 8.0, 10 mM MgCl2, 10 mM 2-mercaptoethanol]. The labeled oligonucleotides were annealed in annealing buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT) starting at 95 °C for 5 min, after which the room temperature was gradually lowered. The oligonucleotides were then purified by a Sephadex G50 column. The binding reaction mixtures (in 25 µL) with in vitro-translated proteins contained 10 mM HEPES-KOH (pH 7.8), 5 mM MgCl2, 1 mM EDTA, 50 mM KCl, 1 mM DTT, 10% Glycerol, 1 µg poly[d(I-C)], protease inhibitor (0.57 mM PMSF, 1 µg/µL Aprotinin, 1 µg/µL Leupeptin and 10 mM Benzamidine) and 3 µL of TNT lysate. The binding reaction mixtures with TNF-treated HeLa cell nuclear proteins contained 10 mM HEPES-KOH pH 7.8, 100 mM KCl, 1 mM EDTA, 10% Glycerol, 0.5 mM DTT, 1 µg poly[d(I-C)], protease inhibitor (0.57 mM PMSF, 1 µg/µL Aprotinin, 1 µg/µL Leupeptin and 10 mM Benzamidine) and 5 µL of nuclear extracts. In the competition experiments, a 10-, 20-, 50- or 500-fold excess of the competing oligonucleotide was supplied before adding the 32P-labeled probe (10 pmol). After incubation at room temperature for 30 min, the samples were electrophoresed in 4.0% acrylamide gels in 0.25 x TAE/2.5% Glycerol. The gels were dried and then exposed to X-ray film for 6–8 h at –80 °C. The oligonucleotides used in these assays are listed in Table 2.
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HeLa S3 cells and 293t cells were maintained in Dulbeccos’ modified Eagles's medium with 10% foetal bovine serum (Hyclone Laboratory, Logan, UT, USA) including 100 U/mL penicillin and 100 µg/mL streptomycin. HeLa and 293t cells were maintained at 37 °C in a 5% CO2 incubator. Transfection was carried out by the standard calcium phosphate method (Invitrogen, San Diego, CA, USA). Briefly, HeLa and 293t cells plated at 50–70% confluency 1 day before transfection were incubated with calcium phosphate-DNA complexes for 24 h at 37 °C. Before extraction for immunoblotting or fixation for immunofluorescence analyses, the cells were cultured in fresh medium for 24 h.
HeLa cells were grown directly on cover slips in 35-mm dishes and then fixed with 100% methanol for 10 min at –30 °C, blocked with 5% goat serum in TBS-T for 1 h at room temperature, then incubated at 4 °C overnight with primary antibody diluted 1 : 300 (anti-Myc antibody (PL14) and anti-HA antibody) or 1 : 100 (anti-KDEL antibody) in TBS-T with 5% goat serum. The cover slips were then washed three times in TBS-T for 5 min, incubated at room temperature for 2 h with an Alexa 488-conjugated goat anti-mouse IgG or Alexa 594-conjugated goat anti-rat IgG (Amersham Pharmacia Biotech) diluted 1 : 300 in 5% goat/TBS-T, and washed three times in TBS-T for 5 min. The cells were counterstained with 0.2 µg/mL DAPI in PBS(-).
For the luciferase assay, HeLa S3 cells were seeded on 24-well plates in DMEM with 10% FBS. Transfection was performed 24 h later using the standard calcium phosphate method (Invitrogen). We transfected HeLa cells with 0.2 µg of pG5 (Promega, Madison, WI) and 0.2 µg of pBIND containing each Tisp40 cDNA. Before extraction for luciferase assay, the cells were cultured in fresh medium for 24 h. 48 h after transfection, the cells were lyzed in passive lysis buffer and assayed for firefly and Renilla luciferase activity by using a Dual-Luciferase reporter Assay system (Promega) according to the manufacturer's instructions. The results were normalized against Renilla luciferase activity. To confirm if Tisp40/ß proteins activate transcription through UPRE, we co-transfected pCMV6Myc Tisp40/ß with pGL3-5XUPRE-luc or pGL3-GRP78(-132)-luc and measured the firefly and Renilla luciferase activities.
Protease protection and Endoglycosidase H assays
For the protease protection assay, transfected HeLa S3 cells were harvested, disrupted using a Dounce-type homogenizer in PBS (-), and then centrifuged at 1000 x g for 5 min to remove the nuclear pellet. The resulting supernatant (9 µg of protein in 90 µL of PBS(-)) was incubated at 37 °C for 15 min with or without varying amounts of trypsin (0, 0.4, 1.2 and 4 µg in 90 µL PBS(-)). Digestion was terminated by adding a quarter volume of 4 x Laemmli's SDS buffer followed by boiling for 10 min. The samples were subjected to SDS PAGE (12% gel), followed by Western blot analysis.
To examine glycosylation of Tisp40/ß, transfected HeLa cell proteins were extracted in RIPA buffer after transfection. The supernatant (25 µg of proteins in 50 µL phosphate-citrate pH 5.0) was incubated overnight at 37 °C with or without 20 mU of Endoglycosidase H, 0.57 mM PMSF, 1 µg/µL Aprotinin, 1 µg/µL Leupeptin and 10 mM Benzamidine. After adding a quarter volume of 4 x Laemmli's SDS buffer, the samples were boiled for 10 min and subjected to SDS PAGE (10% gel) and Western blot analysis.
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Supplementary material |
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http://www.blackwellpublishing.com/products/journals/suppmat/GTC/GTC860/GTC860sm.htm
Figure 1 GFP-Tisp40/ßbZip without NLS could not enter into nucleus. (A) Schematic presentations of Tisp40ß and its truncated construct (Tisp40/ßbZip). (B) Tisp40/ßbZip could not enter into nucleus. Plasmids that can express GFP-Tisp40bZip, GFP-Tisp40ßbZip or GFP alone (a negative control) were transfected into HeLa cells. Then, the cells were counterstained with DAPI (blue) to visualize the nucleus. Merged images are shown.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: snj-0212{at}biken.osaka-u.ac.jp
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References |
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Received: 1 February 2005
Accepted: 3 March 2005
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