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1 Department of Molecular and Cellular Biology, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan
2 Laboratory of Gene Regulation, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
3 Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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A growing number of proteins have been identified as the phosphorylated CTD-interacting factors and many of them participate in coordinating various nuclear events in metabolism of Pol II-transcribing RNA (Hirose & Ohkuma 2007; Egloff & Murphy 2008). It has been recently shown that several WW domain-containing proteins interact with the phosphorylated CTD through their WW domains both in yeast and human (Sudol et al. 2001; Phatnani & Greenleaf 2006; Hirose & Ohkuma 2007). The WW domains are small protein modules (35–40 amino acids in length) that mediate protein–protein interactions through recognizing proline-rich peptide motifs and are found in more than 50 distinct human proteins. The WW domains are fold into a common three-stranded antiparallel β-sheets and classified into four groups based on their target specificities (Sudol et al. 2001). Group IV WW domains specifically recognize the phosphorylated Ser/Thr-Pro (pS/T-P) motifs created by proline-directed protein kinases including mitogen-activated protein kinases (MAPK) and cyclin-dependent protein kinases (CDK) (Lu & Zhou 2007). The consensus CTD heptad contains two Ser-Pro dipeptides and both serine residues are dynamically phosphorylated by specific CDK and MAPK during transcription cycle, cell cycle and stress responses (Phatnani & Greenleaf 2006). As expected, the phosphorylated CTD was shown to be a target of group IV WW domain-containing proteins such as prolyl isomerase (PPIase) Pin1 in human and Ess1 in yeast (Albert et al. 1999; Morris et al. 1999; Verdecia et al. 2000; Wu et al. 2000).
Recently, we identified a novel human WW domain-containing protein PCIF1 as a phosphorylated CTD-interacting factor (Fan et al. 2003; Hirose et al. 2008). The PCIF1 WW domain (PCIF-WW) exhibits the considerable homology to the WW domain of human Pin1 (Pin1-WW). We previously showed that PCIF1-WW directly interacts with the CTD in a phosphorylation-dependent manner and inhibits CTD dephosphorylation by SCP1 phosphatase in vitro. PCIF1 mRNA is ubiquitously expressed in most human tissues and PCIF1 protein is expressed as an 
80 kDa polypeptide. Co-immunoprecipitation studies revealed that PCIF1 associates with the phosphorylated Pol II in vivo. Immunofluorescence microscopy demonstrated that the endogenous PCIF1 colocalizes with Pol IIO and the transcription elongation factor DSIF in the cell nucleus. We also previously observed that PCIF1 significantly represses the reporter gene expression mediated by various transcriptional activators in human cultured cells (Hirose et al. 2008). Although these data suggest that PCIF1 modulates phosphorylation status of the CTD and negatively regulates gene expression by Pol II, exact cellular functions of PCIF1 remain to be elucidated.
As described above, human Pin1 has been reported to interact with Pol IIO (Albert et al. 1999; Verdecia et al. 2000). Pin1 regulates functions and stabilities of various proteins by catalyzing cis-trans isomerization of the pS/T-P motifs in the targets and has been implicated in diverse cellular processes including cell cycle progression, growth-signal responses and neural function (Lu & Zhou 2007). Using the in vitro assays, Xu et al. previously showed that Pin1 inhibits CTD dephosphorylation by FCP1 phosphatase, Pol II-stimulated pre-mRNA splicing and transcription during the early stage of the transcription cycle (Xu et al. 2003; Xu & Manley 2007). Furthermore, inducible over-expression of Pin1 in mammalian cultured cells leads to increasing global phosphorylation of Pol II and reducing Pol II activities possibly by dissociating Pol II from specific genes and accumulating Pol IIO in speckle-related structures in the cell nucleus (Xu et al. 2003; Xu & Manley 2007). Thus, Pin1 appears to play inhibitory roles in transcription specifically at the initiation stage by binding and isomerizing the phosphorylated CTD. On the contrary, two other in vivo studies have presented the apparently controversial observations; one was that the promoter-tethered Pin1 could rather activate the reporter gene expression by Pol II (Komuro et al. 1999), and another was that Pin1 over-expression has no effect on reporter gene expression under control of the cytomegalovirus promoter (Chao et al. 2001). Furthermore, Ess1 (budding yeast orthologue of mammalian Pin1) appears to play positive roles in transcriptional activation by Pol II (Wilcox et al. 2004; Krishnamurthy et al. 2009). Thus, the functions of Pin1 in regulating Pol II activities remain controversial.
In the present study, we first investigated cellular functions of PCIF1 by gene targeting using chicken B-cell DT40 lines. The cell proliferation and the cell cycle progression of DT40 cells were not significantly affected by the disruption of PCIF1 gene. However, we observed up-regulation of Pin1 in the PCIF1-deficient mutant cells. Pin1 activation was also occurred in another independently established PCIF1-deficient cell line, indicating that loss of PCIF1 gene led to the Pin1 over-expression. As reconstitution of the full-length PCIF1 protein in the knockout cells did not reduce Pin1 expression, PCIF1 may not be a negative regulator of Pin1 expression. These results raise the possibility that Pin1 may genetically interact with PCIF1 and that its over-expression may suppress for the loss of PCIF1 in DT40 cells. We furthermore compared these two proteins for their functional properties in human cultured cells. We found that PCIF1 and Pin1 exhibit closest target specificity among other CTD-binding WW proteins, overlapping subcellular localization and comparative inhibitory effects on transcriptional activation by Pol II. These results suggest that Pin1 has some overlapping functions with PCIF1 in vertebrate cells.
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Results |
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To investigate the cellular function of PCIF1, we generated PCIF1-deficient chicken B-cell DT40 lines by gene targeting. We first cloned and sequenced a cDNA encoding a full-length chicken PCIF1. The chicken PCIF1 protein consists of 707 amino acids and exhibits 86% identity to the human PCIF1 protein overall. To prepare targeting constructs, we amplified a genomic DNA fragment covering from the third to 11th exons of the PCIF1 gene by PCR from DT40 genomic DNA and inserted drug-resistant cassettes into the fourth exon of the fragement to disrupt the coding capacity (Fig. 1a). As there are three alleles of the PCIF1 gene in the DT40 genome, we sequentially introduced three kinds of knockout constructs into the parental wild-type DT40 cell line (tWT) that constitutively expresses the Tet repressor fused to the herpes simplex virus VP16 activation domain (tetR-VP16). Southern blot analysis demonstrated that two alleles (lane 2) and all three alleles (lane 3) of the PCIF1 gene were successfully targeted (Fig. 1b). Consequently, we isolated one homozygous mutant clone by screening 54 drug-resistant clones derived from the heterozygous mutants. PCIF1 expression was reduced in the heterozygous mutant tB7+/–/– (lane 2) and completely lost in the homozygous mutant tH8–/–/– (lane 3) at RNA (Fig. 1c) and protein levels (Fig. 1d). Successful establishment of the knockout mutant indicates that PCIF1 is not essential for cell viability at least under normal culture condition. We next compared the mutant and wild-type DT40 cells for their growth properties. The growth curves of the mutant and wild-type DT40 cells exhibited no significant difference (Fig. 1e). We also compared both cell lines for the cell cycle distribution by fluorescence-activated cell sorting (FACS) analysis. There was no significant difference in the cell cycle distribution profile between these two cell lines (data not shown). These observations suggest that PCIF1 does not essentially participate in cell proliferation and cell cycle progression under normal culture condition.
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We next asked whether the loss of PCIF1 might affect gene expression in DT40 cells. We compared the homozygous mutant cells and the wild-type cells for expression levels of several chicken genes (data not shown). Interestingly, we found that chicken prolyl isomerase Pin1 is notably up-regulated in the knockout cells. As shown in Fig. 2a, the mRNA level of Pin1 in the homozygous mutant is markedly increased in the knockout cells compared with the wild-type cells. Immunoblotting results clearly demonstrated that the protein level of Pin1 was also significantly enhanced in the PCIF1-deficient cells (Fig. 2b). As we could isolate and characterize the only one homozygous mutant clone in this targeting experiment, we attempted to independently establish other PCIF1-deficient clones and examined whether Pin1 expression will also be increased in other knockout cells. We again carried out the three rounds of gene targeting and successfully established a new homozygous mutant clone (H1–/–/–) from the wild-type DT40 cells (WT+/+/+). We then compared the both cells for the expression level of Pin1 mRNA by real-time RT-PCR. Two-fold up-regulation of Pin1 was observed in this new knockout clone (compare WT and H1 in Fig. 2c). The protein level of Pin1 was also increased (Fig. 2d, middle, compare lane 1 and 2). These results excluded the possibility that the Pin1 elevation might be caused by an occasional change of gene expression pattern independent of PCIF1 presence in the process of establishing a knockout clone.
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We previously reported that inducible over-expression of Pin1 in mammalian cells resulted in global enhancement of CTD phosphorylation (Xu et al. 2003). Because Pin1 expression was increased in the PCIF1-deficient DT40 cells as shown above, we examined whether Pol II will get global enhancement of CTD phosphorylation in the mutant clones. Immunoblotting using phosphorylation-specific CTD antibodies demonstrated that the global phosphorylation status of the CTD in the both homozygous mutant cell lines, tH8–/–/– (Fig. 3a) and H1–/–/– (Fig. 3b), was not significantly changed when compared with the wild-type cell lines (pSer2 and pSer5).
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The above observations raise the possibility that Pin1 and PCIF1 may have some overlapping functions in vertebrate cells. Hence, we furthermore compared these two proteins for their functional properties. We first compared WW domains derived from the various reported CTD-binding proteins for their specificities and affinities to the phosphorylated CTD. Figure 4a shows a multiple sequence alignment of the WW domains used in this work, including human PCIF1 (Fan et al. 2003), human Pin1 (Albert et al. 1999), yeast Ess1 (Morris et al. 1999), human PQBP-1 (polyglutamine tract binding protein-1)/Npw38 (Okazawa et al. 2002), human YAP (yes-associated protein) (Gavva et al. 1997), human orthologue of yeast splicing factor PRP40 (Morris & Greenleaf 2000), mouse E3 ubiquitin-protein ligase Nedd4 (Gavva et al. 1997; Beaudenon et al. 1999), human WW domain-containing oxidoreductase WWOX (Ingham et al. 2005) and human interferon stimulated gene product ISG95/KIAA0082 (Haline-Vaz et al. 2008). We performed a blot overlay assay in which equivalent amount of each glutathione S-transferase (GST) fused WW domains were probed with 32P-labeled phosphorylated full-length mouse CTD. To reduce the signal that might be derived from the interaction between each WW domain and unphosphorylated portion of the CTD, we added a 10-fold excess of cold unphosphorylated CTD to the binding reaction. As shown in Fig. 4b, the WW domains of PCIF1, Pin1 and Ess1 strongly bound to the phosphorylated CTD (lanes 1–3), whereas other WW domains and the control GST exhibited very low or undetectable binding affinities for the phosphorylated CTD (lanes 4–11). Noteworthy, although the WW domains of hYAP and mNedd4 were reported to have significant binding affinities to the CTD in a similar blot overlay assay (Gavva et al. 1997), both of these failed to bind to the phosphorylated CTD (lanes 7 and 9). Thus, our binding condition is suitable for detecting preferential binding to the phosphorylated CTD over the unphosphorylated CTD. The WW domains of hPQBP-1/Npw38 and hPRP40 are also expected to show significant binding to the phosphorylated CTD with the reported results (Morris & Greenleaf 2000; Okazawa et al. 2002). However, these WW domains did not efficiently bind to the phosphorylated CTD in our assay condition (lanes 4–6).
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Recently, the WW domains of other human proteins such as WW domain-containing oxidoreductase hWWOX and interferon-stimulated gene product ISG95/KIAA0082 have been reported to interact with Pol II (Ingham et al. 2005; Haline-Vaz et al. 2008). We examined whether these WW domains can interact with Pol IIO by GST pull-down assays. In these assays, we also used mitotic cell extracts from HeLa cells treated with nocodazole to accumulate hyperphosphorylated form of Pol II that is preferential target of Pin1 (Lu et al. 1999; Xu et al. 2003). As shown in Fig. 4d, not only PCIF1 and Pin1 WW domains, but also the first WW domain of hWWOX (hWWOX-WW1) efficiently precipitated Pol IIO from untreated HeLa cell extracts (lanes 5, 7 and 9, upper panel). As expected, larger amounts of Pol IIO were precipitated from the nocodazole-treated mitotic extracts by these WW domains (lanes 6, 8 and 10, upper). However, PCIF1-WW mutant (S54H) (Hirose et al. 2008) and ISG95-WW much less efficiently precipitated Pol IIO (lanes 3, 4, 11 and 12, upper). This is the first observation that tumor suppressor candidate gene product hWWOX can associated with the phosphorylated Pol II.
It has been reported that Pin1 interacts with human transcription elongation factor Spt5 (hSpt5) phosphorylated by P-TEFb in vitro and that the hSpt5 CTR domain, which resembles the CTD heptad, is required for interaction (Lavoie et al. 2001; Yamada et al. 2006). Thus, we tested whether hSpt5 might also be precipitated in the GST pull-down assays. As shown in Fig. 4d, Pin1 and PCIF-WW but not other WW domains significantly interacted with the endogenous hSpt5 that may be hyperphosphorylated in mitotic cell extracts (lower panel, lanes 6 and 8).
These all results suggest that PCIF1-WW most closely resembles Pin1-WW for the target recognition specificity among the reported CTD-binding WW domains.
Pin1 partially colocalizes with PCIF1 in the cell nucleus
Our previous study demonstrated that endogenous PCIF1 mainly localizes to the nucleoplasm and colocalizes with Pol IIO and hSpt5 in human cultured cells (Hirose et al. 2008). Although transiently expressed Pin1 proteins have been reported to primarily localize to the nucleus, especially accumulate in SC35-containing nuclear fosi (speckle) (Lu et al. 1996), subcellular localization of endogenous Pin1 has not been extensively analyzed in relation to Pol IIO localization. Thus, we compared the localization of endogenous Pin1, Pol IIO and PCIF1 in human cultured cells by confocal immunofluorescence microscopy. As shown in Fig. 5, endogenous Pin1 was substantially colocalized with Ser2- (A), Ser5- (B) phosphorylated Pol IIO and endogenous PCIF1 (C). These results support the idea that PCIF1 and Pin1 share some nuclear targets including Pol IIO and possibly hSpt5.
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We previously reported that PCIF1 negatively modulates the reporter gene expression by Pol II in human cultured cells (Hirose et al. 2008). Recent studies using an in vitro transcription system have shown that Pin1 also negatively regulates the Pol II activities during the early stage of the transcription cycle (Xu & Manley 2007). However, two other in vivo studies using mammalian cultured cells have presented the different effects of Pin1 on the reporter gene expression (Komuro et al. 1999; Chao et al. 2001). To address the issue whether Pin1 and PCIF1 share some functions in gene expression by Pol II, we tested how Pin1 modulates Pol II activity by using the same reporter assay we previously employed for the study on PCIF1 function (Hirose et al. 2008). We initially examined the effects of Pin1 and PCIF1 over-expression on reporter gene expression activated by various transcriptional activation domains (TAD). As shown in Fig. 6a, over-expression of PCIF1 or Pin1 strongly repressed the VP16-mediated transactivation of the reporter gene, whereas the WW domain mutant Pin1 [Pin1(Y23A)] partially compromised the inhibitory effect. Likewise, marked and similar inhibitory effect of PCIF1 or Pin1 on the transactivations by the TAD of cellular transcription factor Elk1 was observed (Fig. 6b). These results suggest that Pin1, like PCIF1, can inhibit transcriptional activation by Pol II.
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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In the present study, we generated PCIF1-deficient chicken DT40 cell lines and observed significant up-regulation of chicken Pin1 in PCIF1-knockout DT40 cells. As Pin1 elevation was observed in two independently established PCIF1-deficient clones, it seems unlikely that Pin1 might be incidentally activated independent of PCIF1 presence in the process of establishing a knockout cell line. However, reconstitution of the full-length PCIF1 protein in the knockout cells did not reverse Pin1 expression. At present, the reason for this failure of reducing Pin1 expression by PCIF1 reconstitution is unclear whereas we cannot exclude the possibility that specific isoforms of cPCIF1 might participate in the down-regulation of cPin1. We speculate that Pin1 might genetically interact with PCIF1 in vertebrate cells and that its over-expression may counteract the loss of PCIF1 protein in DT40 cells. Global gene expression profiling of the wild-type and knockout mutant cells by DNA microarray analysis and PCIF1-Pin1 double knockout experiment will help us to unravel the reason for the Pin1 elevation by PCIF1 deficiency.
Pin1 has been originally identified as a cell cycle regulator and subsequently reported to participate in various other cellular processes including transcriptional regulation, neural function and various responses against growth-signal, stress and immunity (Lu & Zhou 2007). Thus, Pin1 over-expression is supposed to pleiotropically affect cellular processes. Our previous results demonstrated that inducible over-expression of Pin1 in mammalian cells increases global phosphorylation of Pol II (Xu et al. 2003). However, in the present study, we observed that global phosphorylation status of Pol II was not significantly altered in the Pin1-elevated PCIF1-deficient cells (Fig. 3). One possible explanation for these different outcomes between mammalian cell lines and the DT40 cells may be that global enhancement of the CTD phosphorylation by Pin1 over-expression might be mammalian-specific phonomenon or that the loss of PCIF1 might counteract the effects of Pin1 over-expression on the CTD phosphorylation status. We favor the latter possibility because we previously observed that PCIF1, like Pin1, inhibits CTD dephosphorylation by the CTD phosphatase SCP1 in vitro (Hirose et al. 2008). We assume that Pin1 and PCIF1 may have redundant cellular functions including roles in regulating the CTD phosphorylation status and Pol II activities.
PCIF1 and Pin1 share target specificity
Using a blot overlay assay, we compare the several reported CTD-binding WW domains for their affinity to the phosphorylated CTD and found the significant interaction between the phosphorylated CTD and the WW domains of PCIF1, Pin1 and ESS1 but not the other WW domains we studied (Fig. 4b). Although the second WW domain of hPRP40 (hPRP40-WW2) did not efficiently bind to the phosphorylated CTD in blot overlay assay (Fig. 4b), it surprisingly precipitated Pol IIO from cell extracts as efficiently as PCIF1 and Pin1 did (Fig. 4c). There are two possible explanations for this result; one is that hPRP40-WW2 may directly recognize endogenous Pol IIO more efficiently than the recombinant phosphorylated GST-CTD or the other is that hPRP40-WW2 may indirectly interacts with endogenous Pol IIO. We favor the latter possibility because we observed that hPRP40-WW2 much less efficiently interacted with the purified Pol IIO complex than PCIF1 did in GST pull-down assays (data not shown). Human PRP40 has been found as a component of early spliceosomal complexes and implicated in the recruitment of the human U1 snRNP to the pre-mRNA or in communication between U1 and U2 snRNPs (Behzadnia et al. 2007). Our observation of the stable interaction between hPRP40-WW2 and Pol IIO in cell extracts implies that hPRP40 may play a role in coordinating transcription with pre-mRNA splicing in mammalian cells.
Recently, WW domains of the human interferon-regulated ISG95/KIAA0082 protein have been reported to interact with Pol II by yeast two-hybrid assay (Haline-Vaz et al. 2008). Loop 1 of ISG95-WW is longer than that of usual WW domains and contains two typical consecutive side chains Ser-Arg/Lys (Fig. 4a, underlined). Both of these structural features are conserved in Pin1-WW family and PCIF1-WW (Verdecia et al. 2000). Thus, ISG95-WW appears to be classified into the class IV WW domain which recognizes the pS/T-P motif. However, as shown in the present study, ISG95-WW much less efficiently precipitated Pol IIO than PCIF1 and Pin1 did, reflecting that their interaction may not be stable enough for the GST pull-down assays we carried out. This result suggests that two consecutive side chains Ser-Arg/Lys in loop 1 may not be sufficient enough for recognizing the pS/T-P motif and probably other side chains or structural features such as previously proposed ‘flexibility’ may be required for the specificity (Peng et al. 2007).
We also observed that both Pin1-WW and PCIF1-WW significantly interacted with the hyperphosphorylated hSpt5 in mitotic cell extracts (Fig.4d). As the first WW domain of hWWOX did not precipitated the hyperphosphorylated hSpt5 despite its association with Pol IIO, hSpt5 might not be indirectly precipitated by PCIF1-WW through mediating interaction between Pol IIO and PCIF1-WW. Our present result is in good agreement with our previous cytological observation that endogenous PCIF1 colocalize with hSpt5 in the cell nucleus (Hirose et al. 2008). It will be interesting to test whether PCIF1 may directly interact with the phosphorylated hSpt5.
Pin1 and PCIF1 similarly repress the transcription activity of Pol II
Recent studies on the structures of the several phosphorylated CTD-binding domains bound to the differentially phosphorylated CTD peptides have revealed that the phosphorylated CTD can adapt different conformations depending on its binding partner, probably through an induced fit mechanism (Meinhart et al. 2005). In all structures of these domains, only the trans-conformations of Pro3 and Pro6 in the CTD heptad were observed in the complex, indicating that proline isomerization may be critical for regulating CTD structure and functions (Meinhart et al. 2005). In this respect, the phosphorylated CTD-recognizing PPIase Pin1 likely plays crucial roles in regulating Pol II activities (Hirose & Manley 2000).
Recently, human Pin1 has been reported to negatively regulate Pol II activity during cell cycle and transcription cycle (Xu et al. 2003; Xu & Manley 2007). Nonetheless, two other studies have presented the apparently different effects of Pin1 on transcription (Komuro et al. 1999; Chao et al. 2001). In the present study, we examined the role of Pin1 in transcriptional activation processes using the same reporter assay we previously employed for the study on PCIF1 function (Hirose et al. 2008). We found that Pin1 intrinsically represses transcription activity by Pol II to a similar extent as PCIF1 does (Fig. 6). The apparent discrepancies between our results and the previous studies may reflect differences in the experimental systems. Our siRNA-mediated knockdown experiments could reveal more physiological function of Pin1 than the previous studies in which effects of Pin1 over-expression were only examined (Komuro et al. 1999; Chao et al. 2001).
By our experimental systems, we are currently able to neither specify the stages of transcription cycle negatively regulated by Pin1 and PCIF1 nor address whether the PPIase activity of Pin1 is required for the inhibitory role in transcription. However, according to the previous reports, Pin1 likely inhibits the early stage of transcription cycle and the PPIase activity seems necessary for its repressive function (Xu et al. 2003; Xu & Manley 2007). In this regard, it is important to test which stages of transcription cycle are negatively modulated by PCIF1 and whether PCIF1 might have the PPIase activity or tightly associate with other PPIase enzymes, yet PCIF1 itself does not appear to possess a typical PPIase domain.
Recent genome-wide chromatin immunoprecipitation studies have demonstrated that Pol II initiates transcription without completion but pauses at promoter proximal region of a wide range of metazoan genes (Guenther et al. 2007; Muse et al. 2007; Zeitlinger et al. 2007). A variety of factors and mechanisms are supposed to be involved in the promoter-proximal pausing of Pol II in higher eukaryotes (Gilmour 2009). Our present studies together with the previous reports (Xu & Manley 2007; Hirose et al. 2008) raise the interesting possibility that PCIF1 and Pin1 might play important roles in the transcriptional pausing by negatively modulating Pol II activity near the promoters. Whereas Pin1 is evolutionarily conserved from yeast to humans, PCIF1 presents only in higher organisms including fly but not in worm and yeast (Hirose et al. 2008). We assume that PCIF1 might arise to perform additional negative regulation of Pol II activity during metazoan evolution.
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Experimental procedures |
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The genomic DNA fragment covering from the third to 11th exons of the chicken PCIF1 gene was amplified from DT40 genomic DNA by long-range PCR with the primers based on the chicken PCIF1 cDNA sequence and cloned into a pBluescript (Stratagene). The puromycin, blasticidin S or hygromycin resistance selection marker genes under the control of the chicken β-actin promoter were inserted into the fourth exon to disrupt the coding capacity. The full-length chicken PCIF1 cDNA was reconstructed from an EST clone encoding partial chicken PCIF1 (a gift of Dr Jean-Marie Buerstedde) and a 5' part of PCIF1 cDNA (obtained by 5'-RACE method) and cloned into pZeoSV2 (Invitrogen) to create pZeo-cPCIF1. cDNA encoding a full-length Pin1 and WW domain of PRP40, WWOX and ISG95 were amplified by PCR from cDNA pool synthesized from HeLa cell total RNA. The human Pin1 cDNA was cloned into pcDNA3 (Invitrogen). The PCR amplified cDNA encoding human WW domains were cloned into pGEX6p-3 (GE Healthcare). The plasmid for expressing Pin1 mutant (Y23A) was prepared as described (Xu et al. 2003). Preparations of the plasmids expressing GST fused WW domains derived from yeast ESS1, mouse Nedd4, human YAP1 and PQBP-1/Npw38 were as described (Gavva et al. 1997; Komuro et al. 1999). All sequences of cDNA inserts derived from PCR amplification were verified by DNA sequencing. Sequences of all primers used in the present study are available upon request.
Cell culture and transfection
DT40 cells were maintained in RPMI 1640 (Nissui) medium supplemented with 10% fetal bovine serum, 1% chicken serum, 50 µM 2-mercaptoethanol, penicillin, streptomycin and 2 mM L-glutamine at 39 °C in a humidified 5% CO2 incubator. Transfections were carried out by electroporation using a GENE Pulser II (Bio-Rad) at 25 µF and 550 V. Drug-resistant clones were selected with medium containing 1.5 mg/mL G418, 25 µg/mL blasticidin S, 0.5 µg/mL puromycin, 2.5 mg/mL hygromycin and 0.4 mg/mL Zeocin. Human 293T and HeLa-S3 cells were maintained as described (Hirose et al. 2008).
Generation of mutant DT40 cells
Wild-type DT40 cells were transfected with pTet-off (Clontech) vector and drug-resistant clones were selected by culturing in G418-containing medium. One of the drug-resistant clones (tWT) constitutively expressing tetR-VP16 protein was selected and used for generating the first PCIF1-deficient DT40 cell line (tH8). Second PCIF1 knockout cell line (H1) was established from wild-type DT40 cell line (WT). Three kinds of linearized targeting construct were sequentially introduced into the parental DT40 cell line (tWT). At each step of gene disruption, genomic DNA was isolated from drug-resistant clones and subjected to Southern blotting and genomic PCR to confirm homologous recombination. To establish stable clones expressing cPCIF1, the pZeo-cPCIF1 was transfected into the homozygous mutant. Zeocin-resistant clones were isolated and screened the PCIF1-expressing clones by immunoblotting.
Southern blotting
Genomic DNA isolated from drug-resistant clones was digested with EcoRI and XbaI, and the resulting fragments were electrophoresed in 1.0% agarose gels and transferred to nylon membranes. Blots were hybridized with a 32P-labeled 5' DNA probe (see Fig. 1a) and analyzed by autoradiograph.
Growth curve and cell cycle distribution analysis
5 x 103 cells were seeded in 12-well plate in triplicate and cultured under normal condition during four days. At each day, concentrations of the live cells were determined with a hematocytometer. For examining cell cycle distribution, 1 to 5 x 106 cells were fixed in 70% ethanol, treated with RNase A and stained with propidium iodide. DNA contents were measured by a FACS Calibur and cell cycle profile were analyzed by Cell Quest software (Becton Dickinson).
Immunoblotting and antibodies
Immunoblotting was performed as described previously (Hirose et al. 2008). The antibodies used were as follows: phosphorylation-specific anti-CTD [H5 (pSer2), H14 (pSer5) and 8WG16 (CTD)] (Covance), anti-Rpb1 (ARNA-3, PROGEN), anti-β-actin (Sigma), [anti-PP1, anti-exportin-1] (Transduction), anti-Pin1 (Upstate, Cell Signaling and Calbiochem). Affinity-purified anti-PCIF1 antibodies were described previously (Hirose et al. 2008).
Northern blotting and RT-PCR
Total RNA was isolated from the wild-type and mutants DT40 cells using RNeasy mini kit (Qiagen). Ten microgram of total RNA was separated in a 1.2% formaldehyde agarose gel and transferred to a nylon membrane. Hybridization of the membrane with the indicated 32P-labeled cDNA probe was carried out as described (Fan et al. 2003). First strand cDNA was synthesized from total RNA by Superscript III reverse transcriptase (Invitrogen) with random hexamer. The indicated cDNA were amplified by PCR with specific primers and analyzed by either agarose gel electrophoresis followed by staining with SYBR Green I (Invitrogen) or quantitative real-time PCR using the SYBR Premix ExTaq (Takara) and the Mx3000P Real-Time PCR System (Stratagene).
Blot overlay assay and GST pull-down assay
All GST-fused recombinant proteins were expressed in Escherichia coli and purified as described previously (Hirose & Manley 1998; Hirose et al. 2008). Protein concentration was determined by Bradford method using bovine serum albumin as a standard. Purified GST-PCIF1-WW was labeled with [
-32P] ATP using heart muscle kinase (Sigma). Unincorporated nucleotides were removed by Sephadex G-50 microspin column (GE Healthcare). Blot overlay assays were performed as described previously (Fan et al. 2003). GST pull-down assay with GST-WW proteins from 293T and HeLa whole cell extracts was performed as described previously (Fan et al. 2003).
Immunofluorescence microscopy
Immunostaining of cells was performed as essentially described (Hirose et al. 2008). Briefly, Huh7 or HeLa cells grown on a coverslip were fixed using 3% paraformaldehyde and treated with 0.5% Triton X-100 for 15 min. After blocking, cells were incubated for 12 h at 4 °C with anti-Pin1 mouse mAb G-8 (Santa Cruz) (1 : 200 dilution) and either phosphorylation-dependent anti-CTD mouse monoclonal antibodies H5 (Covanc) (1 : 100 dilution) or 8A7 (Transduction) (1 : 100 dilution), or affinity-purified anti-PCIF1 rabbit polyclonal antibodies (1 : 200 dilution). Reacting antibodies were stained with Alexa Fluor 488-conjugated goat anti-mouse IgG and either with Alexa Fluor 568-conjugated goat anti-rabbit IgG or anti-mouse IgM (Invitrogen). Fluorescence microscopy of fixed cells was performed using a Leica TCS SP5 laser scanning confocal microscope.
Luciferase reporter assays
Luciferase reporter assays to determine the effect of either the over-expression or siRNA-mediated knockdown of PCIF1 or Pin1 on reporter gene activation in human cell lines were performed as described previously (Hirose et al. 2008). Four double-stranded RNA oligonucleotides (NC, PCIF1, Pin1–1 and Pin1–2) were designed and synthesized by Invitrogen. The sequences of siRNA are available upon request.
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Acknowledgements |
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Footnotes |
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These authors contributed equally to this work.
* yh620{at}pha.u-toyama.ac.jp |
References |
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Received: 4 June 2009
Accepted: 30 June 2009
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