VOLUME 288 (2013) PAGES 4522–4537 In Fig. 6B, the wrong image was used for siFam57b-B. This error has been corrected with the correct image and does not affect any results or conclusions of this work.
Positive cofactor 4 (PC4), originally identified as a transcriptional coactivator, possesses the ability to suppress promoter-driven as well as nonspecific transcription via its DNA binding activity. Previous studies showed that the repressive activity of PC4 on promoter-driven transcription is alleviated by transcription factor TFIIH, possibly through one of its enzymatic activities. Using recombinant TFIIH, we have analyzed the role of TFIIH for alleviating PC4-mediated transcriptional repression and determined that the excision repair cross complementing (ERCC3) helicase activity of TFIIH is the enzymatic activity that alleviates PC4-mediated repression via β-γ bond hydrolysis of ATP. In addition, the alleviation does not require either ERCC2 helicase or cyclin-dependent kinase 7 kinase activity. We also show that, as complexed within TFIIH, the cyclin-dependent kinase 7 kinase does not possess the activity to phosphorylate PC4. Thus, TFIIH appears to protect promoters from PC4-mediated repression by relieving the topological constraint imposed by PC4 through the ERCC3 helicase activity rather than by reducing the repressive activity of PC4 via its phosphorylation. Positive cofactor 4 (PC4), originally identified as a transcriptional coactivator, possesses the ability to suppress promoter-driven as well as nonspecific transcription via its DNA binding activity. Previous studies showed that the repressive activity of PC4 on promoter-driven transcription is alleviated by transcription factor TFIIH, possibly through one of its enzymatic activities. Using recombinant TFIIH, we have analyzed the role of TFIIH for alleviating PC4-mediated transcriptional repression and determined that the excision repair cross complementing (ERCC3) helicase activity of TFIIH is the enzymatic activity that alleviates PC4-mediated repression via β-γ bond hydrolysis of ATP. In addition, the alleviation does not require either ERCC2 helicase or cyclin-dependent kinase 7 kinase activity. We also show that, as complexed within TFIIH, the cyclin-dependent kinase 7 kinase does not possess the activity to phosphorylate PC4. Thus, TFIIH appears to protect promoters from PC4-mediated repression by relieving the topological constraint imposed by PC4 through the ERCC3 helicase activity rather than by reducing the repressive activity of PC4 via its phosphorylation. positive cofactor 4 excision repair cross-complementing cyclin-dependent kinase preinitiation complex double-stranded DNA single-stranded DNA RNA polymerase II TATA box-binding protein carboxyl-terminal domain nickel-nitrilotriacetic acid transcription factor nucleotide human immunodeficiency virus Positive cofactor 4 (PC4)1 was originally identified in the upstream-factor stimulatory activity that augments activator-dependent transcription in vitro (1Meisterernst M. Roy A.L. Lieu H.M. Roeder R.G. Cell. 1991; 66: 981-993Abstract Full Text PDF PubMed Scopus (225) Google Scholar,2Kaiser K. Meisterernst M. Trends Biochem. Sci. 1996; 21: 342-345Abstract Full Text PDF PubMed Scopus (92) Google Scholar). PC4 stimulates transcription in vitro with diverse kinds of activators, including VP16 (3Kretzschmar M. Kaiser K. Lottspeich F. Meisterernst M. Cell. 1994; 78: 525-534Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 4Ge H. Roeder R.G. Cell. 1994; 78: 513-523Abstract Full Text PDF PubMed Scopus (307) Google Scholar), thyroid hormone receptor (5Fondell J.D. Guermah M. Malik S. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1959-1964Crossref PubMed Scopus (132) Google Scholar), octamer transcription factor-1 (6Luo Y. Ge H. Stevens S. Xiao H. Roeder R.G. Mol. Cell. Biol. 1998; 18: 3803-3810Crossref PubMed Scopus (43) Google Scholar), and BRCA-1 (7Haile D.T. Parvin J.D. J. Biol. Chem. 1999; 274: 2113-2117Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), presumably by facilitating assembly of the preinitiation complex through bridging between activators and the general transcriptional machinery (4Ge H. Roeder R.G. Cell. 1994; 78: 513-523Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 8Kaiser K. Stelzer G. Meisterernst M. EMBO J. 1995; 14: 3520-3527Crossref PubMed Scopus (92) Google Scholar). Studies on the interaction of PC4 with activators and TFIIA, as well as in vitro functional analyses, suggest that interaction between TFIIA and PC4 plays a pivotal role for facilitating the preinitiation complex (PIC) assembly (3Kretzschmar M. Kaiser K. Lottspeich F. Meisterernst M. Cell. 1994; 78: 525-534Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 4Ge H. Roeder R.G. Cell. 1994; 78: 513-523Abstract Full Text PDF PubMed Scopus (307) Google Scholar). Further studies also demonstrated the importance of PC4 for transcriptional activation by AP-2 (9Kannan P. Tainsky M.A. Mol. Cell. Biol. 1999; 19: 899-908Crossref PubMed Scopus (52) Google Scholar) and HIV transactivator Tat (10Holloway A.F. Occhiodoro F. Mittler G. Meisterernst M. Shannon M.F. J. Biol. Chem. 2000; 275: 21668-21677Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) in vivo. In addition, a yeast homologue of PC4, SUB1/TSP1 (11Henry N.L. Bushnell D.A. Kornberg R.D. J. Biol. Chem. 1996; 271: 21842-21847Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 12Knaus R. Pollock R. Guarente L. EMBO J. 1996; 15: 1933-1940Crossref PubMed Scopus (96) Google Scholar), which is essential for viability in the presence of TFIIB mutations (12Knaus R. Pollock R. Guarente L. EMBO J. 1996; 15: 1933-1940Crossref PubMed Scopus (96) Google Scholar), was shown to function as a coactivator for GCN4 and HAP proteins. The N-terminal region of PC4 contains a serine-rich portion termed the SEAC domain, which exhibits similarity to viral immediate-early proteins (3Kretzschmar M. Kaiser K. Lottspeich F. Meisterernst M. Cell. 1994; 78: 525-534Abstract Full Text PDF PubMed Scopus (166) Google Scholar). Phosphorylation of the serine residues in the SEAC domain negatively regulates the coactivator activity of PC4 (3Kretzschmar M. Kaiser K. Lottspeich F. Meisterernst M. Cell. 1994; 78: 525-534Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 13Ge H. Zhao Y. Chait B.T. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12691-12695Crossref PubMed Scopus (64) Google Scholar) possibly by a conformational change. In addition to the role as coactivator, PC4 was subsequently shown to repress promoter-driven transcription as well as nonspecific transcription in vitro (14Werten S. Stelzer G. Goppelt A. Langen F.M. Gros P. Timmers H.T. Van der Vliet P.C. Meisterernst M. EMBO J. 1998; 17: 5103-5111Crossref PubMed Scopus (58) Google Scholar, 15Kershnar E. Wu S.-Y. Chiang C.-M. J. Biol. Chem. 1998; 273: 34444-34453Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The analyses of PC4 mutants demonstrated that the repressive activity is a separate function from the coactivator activity (14Werten S. Stelzer G. Goppelt A. Langen F.M. Gros P. Timmers H.T. Van der Vliet P.C. Meisterernst M. EMBO J. 1998; 17: 5103-5111Crossref PubMed Scopus (58) Google Scholar); therefore, the repressive activity of PC4 may play an as yet unknown function in regulating transcriptionin vivo. In fact, the primary function of PC4 in vivo could possibly be to repress transcription rather than to enhance transcription because phosphorylated PC4, which is inactive as a coactivator but retains repressive activity, is the predominant form (∼95%) within the cells (13Ge H. Zhao Y. Chait B.T. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12691-12695Crossref PubMed Scopus (64) Google Scholar). Transcriptional repression by PC4 correlates with the single-stranded (ss) DNA binding activity present in its C-terminal region, which shows preferential binding to melted double-stranded (ds) DNA and to heteroduplex DNA (14Werten S. Stelzer G. Goppelt A. Langen F.M. Gros P. Timmers H.T. Van der Vliet P.C. Meisterernst M. EMBO J. 1998; 17: 5103-5111Crossref PubMed Scopus (58) Google Scholar). The structural studies show that PC4 forms a homodimer via its C-terminal region that contains four-stranded β-sheets rich in positively charged and aromatic residues involved directly in binding to ssDNA (16Brandsen J. Werten S. van der Vliet P.C. Meisterernst M. Kroon J. Gros P. Nat. Struct. Biol. 1997; 4: 900-903Crossref PubMed Scopus (66) Google Scholar, 17Werten S. Wechselberger R. Boelens R. van der Vliet P.C. Kaptein R. J. Biol. Chem. 1999; 274: 3693-3699Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Interestingly, in contrast to its coactivator activity, the ssDNA binding activity of PC4 is augmented by phosphorylation of its N-terminal region (8Kaiser K. Stelzer G. Meisterernst M. EMBO J. 1995; 14: 3520-3527Crossref PubMed Scopus (92) Google Scholar). Further studies indicate that PC4-mediated repression of specific transcription from promoters is alleviated by TFIIH, possibly through its enzymatic activities that require β-γ hydrolysis of ATP (14Werten S. Stelzer G. Goppelt A. Langen F.M. Gros P. Timmers H.T. Van der Vliet P.C. Meisterernst M. EMBO J. 1998; 17: 5103-5111Crossref PubMed Scopus (58) Google Scholar, 15Kershnar E. Wu S.-Y. Chiang C.-M. J. Biol. Chem. 1998; 273: 34444-34453Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). However, the identity of the enzymatic activity responsible for the alleviation as well as the mechanism by which TFIIH alleviates PC4-mediated repression remains unknown. Here we used the recombinant TFIIH mutants that lack one of the enzymatic activities (cdk7 kinase, ERCC2 helicase, or ERCC3 helicase) (18Fukuda A. Nogi Y. Hisatake K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1206-1211Crossref PubMed Scopus (17) Google Scholar) and examined the mechanism by which TFIIH counteracts the repressive effect of PC4. We have found that TFIIH counteracts PC4-mediated repression via ERCC3 helicase activity and that neither ERCC2 helicase nor cdk7 kinase activity is required for alleviating the repression, an observation further supported by the fact that TFIIH does not phosphorylate PC4. Our results suggest that PC4 and the ERCC3 helicase activity of TFIIH may act together to increase the specificity of transcription and also to provide more intricate regulation of transcription. PC4 was expressed inEscherichia coli, BL21(DE3)pLysS, harboring the plasmid pET11c-PC4, and the extract was prepared by sonication in buffer A (20 mm Hepes-KOH, pH 7.9, 10% glycerol, 1 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 1 mmdithiothreitol containing 100 mm KCl). The extract was applied onto a HiTrap SP column, and the bound proteins were eluted with a 5-column volume of a linear gradient of 0.1–0.6 M KCl. The eluted fractions were diluted to adjust the conductivity to that of 0.1m KCl and then loaded onto a HiTrap heparin column. The bound proteins were eluted with a 5-column volume of linear gradient of 0.1–0.6 M KCl. RNA polymerase II (RNAPII), TFIIB, TFIIE, TFIIF, and FLAG-tagged TBP (f:TBP) were prepared essentially as described (19Fukuda A. Yamauchi J. Wu S.-Y. Chiang C.-M. Muramatsu M. Hisatake K. Genes Cells. 2001; 6: 707-719Crossref PubMed Scopus (20) Google Scholar). Recombinant TFIIH and its mutants were reconstituted in High Five cells using three baculoviruses, each of which expresses three subunits of TFIIH (18Fukuda A. Nogi Y. Hisatake K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1206-1211Crossref PubMed Scopus (17) Google Scholar,19Fukuda A. Yamauchi J. Wu S.-Y. Chiang C.-M. Muramatsu M. Hisatake K. Genes Cells. 2001; 6: 707-719Crossref PubMed Scopus (20) Google Scholar). The purification of TFIIH was done essentially as described (19Fukuda A. Yamauchi J. Wu S.-Y. Chiang C.-M. Muramatsu M. Hisatake K. Genes Cells. 2001; 6: 707-719Crossref PubMed Scopus (20) Google Scholar) except that TALONTM metal affinity resin (Clontech) was used in place of Ni-nitrilotriacetic acid (NTA) superflow (Qiagen). The amount of each TFIIH, whose cdk7 subunit is C-terminal-tagged with a FLAG epitope, was adjusted by using silver-stained gels as well as quantitative immunoblots with anti-FLAG M2 antibody. In vitro transcription reactions were carried out in a 25-μl reaction containing 12 mm Hepes-KOH, pH 7.9, 6% glycerol, 60 mm KCl, 0.6 mm EDTA, 8 mm MgCl2, 5 mm dithiothreitol, 20 units of RNase inhibitor (TaKaRa), 0.2 mm ATP, 0.2 mm UTP, 0.1 mm3′-O-methyl GTP, 12.5 μm CTP, 10 μCi of [α-32P]CTP, 20 ng TFIIA, 10 ng TFIIB, 4 ng f:TBP, 10 ng TFIIE, 20 ng TFIIF, 20 ng recombinant TFIIH, 100 ng RNAPII, and the indicated amount of PC4. All the transcription reactions contained negatively supercoiled pMLΔ53 (100 ng) as a template. The reactions were performed at 30 °C for 1 h, stopped by the addition of 20 mm EDTA, 0.2% SDS, and 5 μg of proteinase K, and further incubated at 37 °C for 1 h. After phenol/chloroform extraction and ethanol precipitation, the transcripts were analyzed by electrophoresis on a 5% denaturing polyacrylamide gel, followed by autoradiography. Phosphorylation of GST-CTD (carboxyl-terminal domain) and PC4 by TFIIH was performed essentially as described (19Fukuda A. Yamauchi J. Wu S.-Y. Chiang C.-M. Muramatsu M. Hisatake K. Genes Cells. 2001; 6: 707-719Crossref PubMed Scopus (20) Google Scholar). Where indicated, casein kinase II (New England Biolabs) was used in place of TFIIH as indicated. To investigate the functional relationship between TFIIH and PC4, we prepared recombinant TFIIH reconstituted in the insect cells infected with three baculoviruses that expressed TFIIH subunits (18Fukuda A. Nogi Y. Hisatake K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1206-1211Crossref PubMed Scopus (17) Google Scholar, 19Fukuda A. Yamauchi J. Wu S.-Y. Chiang C.-M. Muramatsu M. Hisatake K. Genes Cells. 2001; 6: 707-719Crossref PubMed Scopus (20) Google Scholar). In vitrotranscription assays were performed with recombinant TBP, TFIIB, TFIIE, TFIIF, and TFIIH together with RNAPII purified from HeLa cells (Fig.1A), using a linearized pMLΔ53C2AT template that contained the adenovirus major late promoter fused with a 380-bp G-less cassette (19Fukuda A. Yamauchi J. Wu S.-Y. Chiang C.-M. Muramatsu M. Hisatake K. Genes Cells. 2001; 6: 707-719Crossref PubMed Scopus (20) Google Scholar). The specific 390-nt transcript was observed only in the presence of all factors. No transcription was observed when one of the factors was omitted from the reaction, indicating that there was no cross-contamination among the factors (Fig. 1B). We next tested whether recombinant TFIIH could alleviate transcriptional repression by PC4. As shown in Fig.2A, even in the absence of TFIIH, the negatively supercoiled template allowed production of the specific 390-nt transcript (lane 1), which was suppressed to less than 5% by the addition of PC4 (lane 2). Adding the increasing amounts of TFIIH, however, gradually restored the levels of transcription (lanes 3–6) to 40–60% of those seen in the absence of both TFIIH and PC4 (lane 1), indicating that recombinant TFIIH can reverse the repressive effect of PC4 in a dose-dependent manner as does natural TFIIH (14Werten S. Stelzer G. Goppelt A. Langen F.M. Gros P. Timmers H.T. Van der Vliet P.C. Meisterernst M. EMBO J. 1998; 17: 5103-5111Crossref PubMed Scopus (58) Google Scholar, 15Kershnar E. Wu S.-Y. Chiang C.-M. J. Biol. Chem. 1998; 273: 34444-34453Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Using the highly purified reconstituted system, we then tested the requirement for β-γ bond hydrolysis by substituting ATP with adenylyl-imidodiphosphate (AMP-PNP) and adenosine-5′-O-(thiotriphosphate) (ATP-γS), both of which can be incorporated into growing RNA chains during transcription but cannot be hydrolyzed at the β-γ bond. When ATP was replaced by non-hydrolyzable AMP-PNP or ATP-γS in the transcription reactions containing both PC4 and TFIIH, virtually no transcription was observed (Fig. 2B, lanes 4 and 6), indicating that β-γ bond hydrolysis of ATP was absolutely required for counteracting PC4-mediated repression. Transcription was restored, however, when AMP-PNP and ATP-γS were further supplemented with dATP (Fig. 2B, lanes 5 and 7), which could provide β-γ bond hydrolysis. These results show the requirement for β-γ bond hydrolysis of ATP (or dATP) for alleviating PC4-mediated repression even in the highly pure transcription system. Because TFIIH is the only known factor that utilizes β-γ bond hydrolysis of ATP in this well defined transcription system, the results clearly demonstrate the involvement of the enzymatic activities of TFIIH in the alleviation. The requirement of β-γ bond hydrolysis suggested that one of the enzymatic activities of TFIIH was required for alleviating the repression by PC4. To determine which enzymatic activity of TFIIH was responsible for the alleviation, we utilized three recombinant TFIIH mutants, each of which is defective in either cdk7 kinase, ERCC3 helicase, or ERCC2 helicase activities (Fig.3, A and B). These mutants have alanine instead of the conserved lysine within the ATP binding site of Walker type A motifs, at the 41st residue of cdk7, 346th residue of ERCC3, and 48th residue of ERCC2, respectively (Fig.3A) (18Fukuda A. Nogi Y. Hisatake K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1206-1211Crossref PubMed Scopus (17) Google Scholar, 20Wada T. Orphanides G. Hasegawa J. Kim D.K. Shima D. Yamaguchi Y. Fukuda A. Hisatake K. Oh S. Reinberg D. Handa H. Mol. Cell. 2000; 5: 1067-1072Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Substitution of the lysine with either arginine or alanine in these motifs is known to eliminate the ability to hydrolyze ATP, resulting in the inactivation of each enzymatic activity. As shown in Fig. 3C, TFIIH with the mutated cdk7 kinase (K41A) and with the mutated ERCC2 helicase (K48A) alleviated PC4-mediated repression as well as wild-type TFIIH, whereas TFIIH with the mutated ERCC3 helicase (K346A) could not alleviate the repression at all. These results demonstrate that ERCC3 helicase activity is the sole enzymatic activity required for alleviating PC4-mediated repression, and neither the cdk7 kinase nor the ERCC2 helicase plays any role in alleviating PC4-mediated repression through ATP hydrolysis. Because the previous result showed that PC4 is released from the template upon phosphorylation by TFIIH (21Malik S. Guermah M. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2192-2197Crossref PubMed Scopus (94) Google Scholar), the dispensability of the cdk7 kinase for PC4-mediated repression was somewhat unexpected. Furthermore, lack of any consensus phosphorylation site for cdks (S/TPXR/K) in PC4 prompted us to re-address whether TFIIH is indeed able to phosphorylate PC4 in vitro as previously reported (15Kershnar E. Wu S.-Y. Chiang C.-M. J. Biol. Chem. 1998; 273: 34444-34453Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 21Malik S. Guermah M. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2192-2197Crossref PubMed Scopus (94) Google Scholar). As shown in Fig. 4A, wild-type TFIIH, K48A, and K346A phosphorylated CTD efficiently but K41A did not phosphorylate CTD, indicating that the substitution of lysine with alanine at the 41st residue of cdk7 eliminated the kinase activity to an undetectable level. Phosphorylation of CTD by TFIIH produced the hypophosphorylated form as well as the hyperphosphorylated form that showed a slower migration on the SDS gel (Fig. 4A). Casein kinase II also phosphorylated CTD, although phosphorylation did not shift the migration of GST-CTD (Fig. 4A, lanes 1and 2). We next tested whether casein kinase II and the same set of TFIIH mutants could phosphorylate PC4. Casein kinase II efficiently phosphorylated PC4 as previously reported (3Kretzschmar M. Kaiser K. Lottspeich F. Meisterernst M. Cell. 1994; 78: 525-534Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 13Ge H. Zhao Y. Chait B.T. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12691-12695Crossref PubMed Scopus (64) Google Scholar) and altered PC4 from the faster migrating form (∼15 kDa) to the slower migrating form (∼20 kDa) (Fig. 4B, bottom panel, lanes 1 and 2). In contrast, wild-type TFIIH, K346A, and K48A, all of which retain cdk7 kinase activity (Fig. 4A,lanes 4, 6, and 7), did not phosphorylate PC4 (Fig. 4B, lanes 4,6, and 7). The low levels of PC4 labeling observed on a longer exposure of the gel (Fig. 4B,middle panel, lanes 3–7) is not because of the TFIIH kinase activity because the TFIIH mutant K41A, which lacks the kinase activity (Fig. 4A, lane 5), showed the same degree of labeling as wild-type TFIIH. Our results demonstrate that TFIIH does not phosphorylate PC4 and argue against the involvement of PC4 phosphorylation by TFIIH for alleviating PC4-mediated repression of transcription. The in vivo concentration of PC4 is estimated to be ∼1 μm in HeLa cells, and ∼95% of PC4 is phosphorylated in vivo, presumably by casein kinase II (3Kretzschmar M. Kaiser K. Lottspeich F. Meisterernst M. Cell. 1994; 78: 525-534Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 13Ge H. Zhao Y. Chait B.T. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12691-12695Crossref PubMed Scopus (64) Google Scholar, 14Werten S. Stelzer G. Goppelt A. Langen F.M. Gros P. Timmers H.T. Van der Vliet P.C. Meisterernst M. EMBO J. 1998; 17: 5103-5111Crossref PubMed Scopus (58) Google Scholar). Therefore, we tested whether phosphorylated and non-phosphorylated PC4 can distinguish the presence and absence of ERCC3 helicase activity within the general transcriptional machinery at the physiological PC4 concentration. PC4 was first phosphorylated by CKII as shown in Fig. 4B, and then increasing amounts of both phosphorylated and non-phosphorylated PC4 were added to the transcriptional reactions containing either wild-type TFIIH or K346A. As shown in Fig.5, the levels of transcription were reduced to less than 40% at 0.125 μm of PC4 and to ∼10% at 1 μm of PC4 in the absence of ERCC3 helicase activity. Non-phosphorylated PC4 repressed transcription slightly better than phosphorylated PC4 in the absence of ERCC3 helicase activity (Fig. 5). By contrast, in the presence of wild-type TFIIH, transcription remained markedly more resistant to repression by PC4 (Fig. 5) (14Werten S. Stelzer G. Goppelt A. Langen F.M. Gros P. Timmers H.T. Van der Vliet P.C. Meisterernst M. EMBO J. 1998; 17: 5103-5111Crossref PubMed Scopus (58) Google Scholar). These results indicate that PC4 represses transcription regardless of its phosphorylation status in the absence of ERCC3 helicase activity. In addition, because repression by PC4 occurs similarly in the presence of K346A (Fig. 5) as in the absence of TFIIH (data not shown) (14Werten S. Stelzer G. Goppelt A. Langen F.M. Gros P. Timmers H.T. Van der Vliet P.C. Meisterernst M. EMBO J. 1998; 17: 5103-5111Crossref PubMed Scopus (58) Google Scholar), mutual exclusion of PC4 and TFIIH on the promoter is an unlikely mechanism for the antagonistic effect of PC4 and TFIIH. Our results show that the ERCC3 helicase activity of TFIIH counteracts PC4-mediated transcriptional repression and that neither the ERCC2 helicase nor the cdk7 kinase has any role in this process. The fact that the ERCC3 helicase, but not the cdk7 kinase, of TFIIH relieves PC4-mediated repression provides a clue as to the mechanism by which TFIIH and PC4 act antagonistically to regulate transcription. Negatively supercoiled templates allow specific transcription by RNAPII in the absence of TFIIH and ATP in vitro (22Parvin J.D. Sharp P.A. Cell. 1993; 73: 533-540Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 23Goodrich J.A. Tjian R. Cell. 1994; 77: 145-156Abstract Full Text PDF PubMed Scopus (287) Google Scholar), presumably by the transfer of free energy stored on the negatively supercoiled templates (24Lilley D.M. Trends Genet. 1988; 4: 111-114Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 25Murchie A.I. Bowater R. Aboul-ela F. Lilley D.M. Biochim. Biophys. Acta. 1992; 1131: 1-15Crossref PubMed Scopus (47) Google Scholar, 26Holstege F.C. Tantin D. Carey M. van der Vliet P.C. Timmers H.T. EMBO J. 1995; 14: 810-819Crossref PubMed Scopus (131) Google Scholar). This transfer of free energy appears to be constrained by PC4, because the property of negatively supercoiled DNA templates bound by PC4 is similar to that of linear DNA templates with regard to the absolute requirement of TFIIH and ATP for specific promoter-driven transcription (22Parvin J.D. Sharp P.A. Cell. 1993; 73: 533-540Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 23Goodrich J.A. Tjian R. Cell. 1994; 77: 145-156Abstract Full Text PDF PubMed Scopus (287) Google Scholar). This effect of PC4 transmitted indirectly through DNA to the general transcriptional machinery is consistent with the functional antagonism between TFIIH and PC4 that does not involve the mutual exclusion of TFIIH and PC4 on the promoter (Fig. 5). Thus, the role for ERCC3 helicase activity may be to overcome the topological constraint conferred by PC4 on negatively supercoiled templates, a process that could potentially prompt the release of PC4 from the promoter region (21Malik S. Guermah M. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2192-2197Crossref PubMed Scopus (94) Google Scholar). Our results, however, rule out the possibility that the cdk7 kinase of TFIIH phosphorylates PC4 (15Kershnar E. Wu S.-Y. Chiang C.-M. J. Biol. Chem. 1998; 273: 34444-34453Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 21Malik S. Guermah M. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2192-2197Crossref PubMed Scopus (94) Google Scholar) and facilitates its release from the promoter (21Malik S. Guermah M. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2192-2197Crossref PubMed Scopus (94) Google Scholar). In light of our study as well as a previous study (14Werten S. Stelzer G. Goppelt A. Langen F.M. Gros P. Timmers H.T. Van der Vliet P.C. Meisterernst M. EMBO J. 1998; 17: 5103-5111Crossref PubMed Scopus (58) Google Scholar), we propose two possible mechanisms by which PC4 represses promoter-independent transcription: i.e. “direct” and “indirect” mechanisms. In the direct mechanism, PC4 binds to ssDNA regions via its ssDNA binding ability, competing directly with RNAPII, and thus physically displaces RNAPII from ssDNA regions (Fig.6A). By contrast, in the indirect mechanism PC4 binds dsDNA regions via its dsDNA binding ability and renders DNA more “rigid” so that the free energy stored in negative superhelicity (24Lilley D.M. Trends Genet. 1988; 4: 111-114Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 25Murchie A.I. Bowater R. Aboul-ela F. Lilley D.M. Biochim. Biophys. Acta. 1992; 1131: 1-15Crossref PubMed Scopus (47) Google Scholar, 26Holstege F.C. Tantin D. Carey M. van der Vliet P.C. Timmers H.T. EMBO J. 1995; 14: 810-819Crossref PubMed Scopus (131) Google Scholar) will not generate transiently melted ssDNA regions that permit RNAPII to initiate random transcription (Fig.6B). It is conceivable that the indirect mechanism provides the primary protection against spurious transcription and the direct mechanism provides a backup. In this scenario, PC4 bound to dsDNA regions may also serve as a reservoir that can be recruited quickly to ssDNA regions where the possibility of spurious transcription is greater. In agreement with the recruitment of PC4 from dsDNA to ssDNA, PC4 binds to ssDNA more strongly than to dsDNA (14Werten S. Stelzer G. Goppelt A. Langen F.M. Gros P. Timmers H.T. Van der Vliet P.C. Meisterernst M. EMBO J. 1998; 17: 5103-5111Crossref PubMed Scopus (58) Google Scholar). PC4-mediated repression of transcription from non-promoter regions as described above may facilitate the efficient allocation of the limiting amount of RNAPII in vivo (27Borggrefe T. Davis R. Bareket-Samish A. Kornberg R.D. J. Biol. Chem. 2001; 276: 47150-47153Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 28Kimura M. Sakurai H. Ishihama A. Eur. J. Biochem. 2001; 268: 612-619Crossref PubMed Scopus (28) Google Scholar), which could be otherwise sequestered onto transiently melted ssDNA regions. In the living cells, DNA is predominantly negatively supercoiled and is also undergoing dynamic topological changes during DNA replication, transcription, and repair, possibly exposing melted ssDNA regions frequently. Spurious transcription from these melted ssDNA regions is likely to be suppressed mainly by phosphorylated PC4, which constitutes ∼95% of PC4 in vivo (13Ge H. Zhao Y. Chait B.T. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12691-12695Crossref PubMed Scopus (64) Google Scholar), because phosphorylated PC4 can strongly suppress promoter-independent (and thus, general transcription factor-independent) transcription from the melted DNA region in vitro (14Werten S. Stelzer G. Goppelt A. Langen F.M. Gros P. Timmers H.T. Van der Vliet P.C. Meisterernst M. EMBO J. 1998; 17: 5103-5111Crossref PubMed Scopus (58) Google Scholar). PC4 may also play a role in preventing spurious transcription from promoters, which in vivo is likely to be negatively supercoiled and from which transcription could be potentially initiated in the absence of TFIIH. When the ERCC3 helicase of TFIIH is active within the general transcriptional machinery, transcription is probably not repressed by PC4 in vivo (Fig. 6C) because the TFIIH ERCC3 helicase activity counteracts the repressive activity of phosphorylated PC4 at the physiological concentration (∼1 μm) (Fig. 5). Indeed, when PC4 is overexpressed in cells in the absence of the HIV transactivator, transcription from the HIV promoter is only marginally reduced or not reduced at all, depending upon the assay conditions (10Holloway A.F. Occhiodoro F. Mittler G. Meisterernst M. Shannon M.F. J. Biol. Chem. 2000; 275: 21668-21677Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). However, if the ERCC3 helicase activity of TFIIH is inhibited (Fig. 5), such as by negative regulator of activated transcription and by FBP interacting repressor (29Akoulitchev S. Chuikov S. Reinberg D. Nature. 2000; 407: 102-106Crossref PubMed Scopus (297) Google Scholar,30Liu J. He L. Collins I. Ge H. Libutti D. Li J. Egly J.M. Levens D. Mol. Cell. 2000; 5: 331-341Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), phosphorylated PC4 may further reduce the low background transcription from promoters even at the physiological PC4 concentration (Fig. 6D). Because TFIIH appears to be sub-stoichiometric (20–30%) to other general transcription factorsin vivo (27Borggrefe T. Davis R. Bareket-Samish A. Kornberg R.D. J. Biol. Chem. 2001; 276: 47150-47153Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), a fraction of PIC might even lack TFIIH and could be repressed by PC4, though this possibility must be rigorously examined in vivo. In any event, regulation of promoter-dependent transcription with a high level of dynamic range in vivo is likely to be contingent upon the presence of PC4, because negatively supercoiled DNA in vivomay permit inadvertent transcription from promoters and could potentially reduce the dynamic range of transcriptional regulation. Several lines of evidence suggest the importance of PC4 in regulating transcription in vivo. First, a yeast homolog of human PC4, SUB4, enhances transcriptional activation by the activators GCN5 and HAP4 (12Knaus R. Pollock R. Guarente L. EMBO J. 1996; 15: 1933-1940Crossref PubMed Scopus (96) Google Scholar), and though PC4 is not essential for viability, its deletion results in inositol auxotrophy, a phenotype observed in the mutations of transcriptional regulators such as SNF/SWI, SRB, and the CTD of RNA polymerase II (31Peterson C.L. Herskowitz I. Cell. 1992; 68: 573-583Abstract Full Text PDF PubMed Scopus (463) Google Scholar, 32Peterson C.L. Dingwall A. Scott M.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2905-2908Crossref PubMed Scopus (340) Google Scholar, 33Thompson C.M. Koleske A.J. Chao D.M. Young R.A. Cell. 1993; 73: 1361-1375Abstract Full Text PDF PubMed Scopus (388) Google Scholar, 34Cairns B.R. Kim Y.J. Sayre M.H. Laurent B.C. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1950-1954Crossref PubMed Scopus (345) Google Scholar). Second, PC4 enhances TAT-dependent transcription from the HIV promoter (10Holloway A.F. Occhiodoro F. Mittler G. Meisterernst M. Shannon M.F. J. Biol. Chem. 2000; 275: 21668-21677Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and restores the reduced AP-2 activity in theras-transformed cell lines by relieving AP-2 self-interference (9Kannan P. Tainsky M.A. Mol. Cell. Biol. 1999; 19: 899-908Crossref PubMed Scopus (52) Google Scholar). Finally, PC4 may play a role as a tumor suppressor in lung and bladder cancers, because the loss of heterogeneity of the PC4 gene is often observed in these cancer cells (35Wieland I. Bohm M. Arden K.C. Ammermuller T. Bogatz S. Viars C.S. Rajewsky M.F. Oncogene. 1996; 12: 97-102PubMed Google Scholar, 36Bohm M. Kirch H. Otto T. Rubben H. Wieland I. Int. J. Cancer. 1997; 74: 291-295Crossref PubMed Scopus (26) Google Scholar). These results demonstrate the importance of PC4 as a regulator of transcription and possibly as a tumor suppressor in vivo. Though the importance of PC4 in vivo has been mainly interpreted in the context of its coactivator activity, the predominance of the repressive form of PC4 in vivo (13Ge H. Zhao Y. Chait B.T. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12691-12695Crossref PubMed Scopus (64) Google Scholar) suggests that some of the observed effects may well be attributed to the reduced precision of transcriptional regulation caused by the loss of the repressive activity of PC4. In conclusion, the repressive activity of PC4 may be essential for the intricate regulation of transcription in conjunction with the ERCC3 helicase of TFIIH. The repressive activity of PC4, and possibly of other ssDNA-binding proteins, may play an important but yet under-appreciated role for more elaborate and fine-tuned regulation of reactions involving DNA molecules. We thank M. Suganuma for technical assistance.
Proper identification of pancreatic ducts is a major challenge for researchers performing partial duct ligation (PDL), because pancreatic ducts, which are covered with acinar cells, are translucent and thin. Although damage to pancreatic ducts may activate quiescent ductal stem cells, which may allow further investigation into ductal stem cells for therapeutic use, there is a lack of effective techniques to visualize pancreatic ducts. In this study, we report a new method for identifying pancreatic ducts. First, we aimed to visualize pancreatic ducts using black, waterproof fountain pen ink. We injected the ink into pancreatic ducts through the bile duct. The flow of ink was observed in pancreatic ducts, revealing their precise architecture. Next, to visualize pancreatic ducts in live animals, we injected fluorescein-labeled bile acid, cholyl-lysyl-fluorescein into the mouse tail vein. The fluorescent probe clearly marked not only the bile duct but also pancreatic ducts when observed with a fluorescent microscope. To confirm whether the pancreatic duct labeling was successful, we performed PDL on Neurogenin3 (Ngn3)-GFP transgenic mice. As a result, acinar tissue is lost. PDL tail pancreas becomes translucent almost completely devoid of acinar cells. Furthermore, strong activation of Ngn3 expression was observed in the ligated part of the adult mouse pancreas at 7 days after PDL.
Although motor coordination or motor skill learning are improved by taking vitamin D in the animal experiment, muscle function have not been estimated. Here we examined the effect of vitamin D3 administration on motor coordination and motor skill learning, muscle strength, and muscle volume in mice fed a vitamin D deficient diet. In mice fed a vitamin D deficient diet, serum calcium and 25(OH)D3 concentrations were measured. We then conducted Rotarod test, beam walking assay, micro-CT analysis, and forelimb grip strength test. Administration of vitamin D3 elongated the retention time in the Rotarod test in a time dependent manner. In contrast, the time to reach a beam goal box in beam walking assay was not changed in mice administered with vitamin D3, compared to the control. Oral administration of vitamin D3 did not affect muscle strength nor muscle volume. Oral administration of vitamin D3 promotes not motor coordination but motor skill learning and does not affect muscle function.
Nax is a sodium-concentration ([Na+])-sensitive Na channel with a gating threshold of ~150 mM for extracellular [Na+] ([Na+]o) in vitro. We previously reported that Nax was preferentially expressed in the glial cells of sensory circumventricular organs including the subfornical organ, and was involved in [Na+] sensing for the control of salt-intake behavior. Although Nax was also suggested to be expressed in the neurons of some brain regions including the amygdala and cerebral cortex, the channel properties of Nax have not yet been adequately characterized in neurons. We herein verified that Nax was expressed in neurons in the lateral amygdala of mice using an antibody that was newly generated against mouse Nax. To investigate the channel properties of Nax expressed in neurons, we established an inducible cell line of Nax using the mouse neuroblastoma cell line, Neuro-2a, which is endogenously devoid of the expression of Nax. Functional analyses of this cell line revealed that the [Na+]-sensitivity of Nax in neuronal cells was similar to that expressed in glial cells. The cation selectivity sequence of the Nax channel in cations was revealed to be Na+ ≈ Li+ > Rb+ > Cs+ for the first time. Furthermore, we demonstrated that Nax bound to postsynaptic density protein 95 (PSD95) through its PSD95/Disc-large/ZO-1 (PDZ)-binding motif at the C-terminus in neurons. The interaction between Nax and PSD95 may be involved in promoting the surface expression of Nax channels because the depletion of endogenous PSD95 resulted in a decrease in Nax at the plasma membrane. These results indicated, for the first time, that Nax functions as a [Na+]-sensitive Na channel in neurons as well as in glial cells.
Abstract The R5 subfamily of receptor-type protein tyrosine phosphatases (RPTPs) comprises PTPRZ and PTPRG. A recent study on primary human glioblastomas suggested a close association between PTPRZ1 (human PTPRZ ) expression and cancer stemness. However, the functional roles of PTPRZ activity in glioma stem cells have remained unclear. In the present study, we found that sphere-forming cells from the rat C6 and human U251 glioblastoma cell lines showed high expression levels of PTPRZ-B, the short receptor isoform of PTPRZ. Stable PTPRZ knockdown altered the expression levels of stem cell transcription factors such as SOX2, OLIG2, and POU3F2 and decreased the sphere-forming abilities of these cells. Suppressive effects on the cancer stem-like properties of the cells were also observed following the knockdown of PTPRG . Here, we identified NAZ2329, a cell-permeable small molecule that allosterically inhibits both PTPRZ and PTPRG. NAZ2329 reduced the expression of SOX2 in C6 and U251 cells and abrogated the sphere-forming abilities of these cells. Tumor growth in the C6 xenograft mouse model was significantly slower with the co-treatment of NAZ2329 with temozolomide, an alkylating agent, than with the individual treatments. These results indicate that pharmacological inhibition of R5 RPTPs is a promising strategy for the treatment of malignant gliomas.
