1: Mol Cell Biol. 2005 Jun;25(11):4638-49. TAF9b (formerly TAF9L) is a bona fide TAF that has unique and overlapping roles with TAF9. Frontini M, Soutoglou E, Argentini M, Bole-Feysot C, Jost B, Scheer E, Tora L. Department of Transcription, Institut de Genetique et de Biologie Moleculaire et Cellulaire, UMR 7104, BP 10142, 67404 Illkirch Cedex, CU de Strasbourg, France. TFIID plays a key role in transcription initiation of RNA polymerase II preinitiation complex assembly. TFIID is comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). A second set of transcriptional regulatory multiprotein complexes containing TAFs has been described (called SAGA, TFTC, STAGA, and PCAF/GCN5). Using matrix-assisted laser desorption ionization mass spectrometry, we identified a novel TFTC subunit, human TAF9Like, encoded by a TAF9 paralogue gene. We show that TAF9Like is a subunit of TFIID, and thus, it will be called TAF9b. TFIID and TFTC complexes in which both TAF9 and TAF9b are present exist. In vitro and in vivo experiments indicate that the interactions between TAF9b and TAF6 or TAF9 and TAF6 histone fold pairs are similar. We observed a differential induction of TAF9 and TAF9b during apoptosis that, together with their different ability to stabilize p53, points to distinct requirements for the two proteins in gene regulation. Small interfering RNA (siRNA) knockdown of TAF9 and TAF9b revealed that both genes are essential for cell viability. Gene expression analysis of cells treated with either TAF9 or TAF9b siRNAs indicates that the two proteins regulate different sets of genes with only a small overlap. Taken together, these data demonstrate that TAF9 and TAF9b share some of their functions, but more importantly, they have distinct roles in the transcriptional regulatory process. PMID: 15899866 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: J Biol Chem. 2004 Oct 8;279(41):42410-21. Epub 2004 Jul 26. ZNF76, a novel transcriptional repressor targeting TATA-binding protein, is modulated by sumoylation. Zheng G, Yang YC. Department of Pharmacology and Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA. Direct interaction of positive and negative regulators with the general transcription machinery modulates transcription. The TATA-binding protein (TBP) is one target for transcriptional regulators. In this study, we identified ZNF76 as a novel transcriptional repressor that targets TBP. ZNF76 interacts with TBP through both its N and C termini, and both regions are required for ZNF76 to exert its inhibitory function on p53-mediated transactivation. The inhibitory effect of ZNF76 on p53 activity was demonstrated by reporter assays and endogenous target gene expression. We mapped the TBP-interacting region in the C terminus of ZNF76 to a glutamic acid-rich domain, which acts in a dominant negative manner to enhance p53-mediated transactivation in reporter assays. Mutagenesis study for ZNF76 suggests a correlation between interaction with TBP and effect on p53-mediated transactivation, supporting the conclusion that ZNF76 targets TBP for transcriptional repression. Chromatin immunoprecipitation experiments suggest that ZNF76 prevents TBP from occupying the endogenous p21 promoter. ZNF76 is sumoylated by PIAS1 at lysine 411, which is in the minimal TBP-interacting region. Overexpression of PIAS1 and SUMO-1 abolishes the interaction between ZNF76 and TBP and partially relieves the repressive effect of ZNF76. These results suggest that ZNF76 functions as a transcriptional repressor through its interaction with TBP and that sumoylation modulates its transcriptional repression activity. Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc. PMID: 15280358 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Mol Cell Biol. 2004 Jun;24(12):5332-9. Activation of a DNA damage checkpoint response in a TAF1-defective cell line. Buchmann AM, Skaar JR, DeCaprio JA. Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA. Although the link between transcription and DNA repair is well established, defects in the core transcriptional complex itself have not been shown to elicit a DNA damage response. Here we show that a cell line with a temperature-sensitive defect in TBP-associated factor 1 (TAF1), a component of the TFIID general transcription complex, exhibits hallmarks of an ATR-mediated DNA damage response. Upon inactivation of TAF1, ATR rapidly localized to subnuclear foci and contributed to the phosphorylation of several downstream targets, including p53 and Chk1, resulting in cell cycle arrest. The increase in p53 expression and the G(1) phase arrest could be blocked by caffeine, an inhibitor of ATR. In addition, dominant negative forms of ATR but not ATM were able to override the arrest in G(1). These results suggest that a defect in TAF1 can elicit a DNA damage response. PMID: 15169897 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Clin Exp Immunol. 2004 Jun;136(3):574-84. Over-expression of TATA binding protein (TBP) and p53 and autoantibodies to these antigens are features of systemic sclerosis, systemic lupus erythematosus and overlap syndromes. Chauhan R, Handa R, Das TP, Pati U. Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, India. The aim of this study was to determine the expression levels of p53 and TATA binding protein (TBP) and the presence of autoantibodies to these antigens in Asian Indian patients with systemic sclerosis (SSc), overlap syndromes (OS) and systemic lupus erythematosus (SLE). Fifty patients with SSc, 20 with OS, including mixed connective tissue diseases (MCTD), 20 with SLE, 10 disease controls (DC) and 25 controls (C) were studied. The over-expression of p53 and TBP antigen was determined quantitatively by sandwich enzyme-linked immunosorbent assay (ELISA), varies between four- and sevenfold higher in patients with SSc, OS and SLE, in comparison to DC and C. The expressed protein antigens were not present as free antigens but as immune-complexes. Autoantibodies to p53 were detected by ELISA in 78% subjects with SSc, 100% with OS and 80% with SLE. Autoantibodies to TBP were observed in 28% patients with SSc, 25% with OS and 15% with SLE. In comparison to healthy controls, the titre of antibodies to p53 was significantly higher in patients with SSc (P = 0.00001) than the patients with OS (P = 0.00279) and SLE (P = 0.00289), whereas the titre of antibodies to TBP was higher in patients with OS (P = 0.00185) than the SLE (P = 0.00673) and the SSc (P = 0.00986) patients. Autoantibodies to p53 and TBP were detected in all these patients and the levels of these two autoantibodies showed weak negative correlation with each other. We propose that the over-expression of these antigens might be due to hyperactive regulatory regions in the p53 and TBP gene. PMID: 15147363 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: J Biol Chem. 2004 Feb 20;279(8):6345-53. Epub 2003 Dec 9. Functional and physical interaction of the human ARF tumor suppressor with Tat-binding protein-1. Pollice A, Nasti V, Ronca R, Vivo M, Lo Iacono M, Calogero R, Calabro V, La Mantia G. Department of Genetics, General and Molecular Biology, University of Naples Federico II, Via Mezzocannone 8, 80134 Naples, Italy. The p14ARF tumor suppressor is a key regulator of cellular proliferation, frequently inactivated in human cancer, whose mode of action is currently not completely understood. We report here that the so-called human immunodeficiency virus Tat-binding protein-1 (TBP-1), a component of the 19 S regulatory subunit of the proteasome 26 S, also involved in transcriptional regulation and with a supposed role in the control of cell proliferation, specifically interacts with ARF, both in yeast and mammalian cells. We present evidence that the overexpression of TBP-1 in various cell lines results in a sharp increase of both transfected and endogenous ARF protein levels. Moreover, this effect depends on the binding between the two proteins and, at least in part, is exerted at the post-translational level. We also show that the ARF increase following TBP-1 overexpression results in an increase in p53 protein levels and activity. Finally, our data underline a clear involvement of TBP-1 in the control of cell proliferation. PMID: 14665636 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Mol Cell Biol. 2003 Oct;23(20):7350-62. Core promoter elements and TAFs contribute to the diversity of transcriptional activation in vertebrates. Chen Z, Manley JL. Department of Biological Sciences, Columbia University, New York, New York 10027, USA. Gene-specific transcriptional activation is a multistep process that requires numerous protein factors and DNA elements, including enhancers and the core promoter. To investigate the roles of core promoter elements in transcriptional activation in vertebrates, we examined expression and factor occupancy on representative promoters in chicken DT40 cells containing a conditional TATA binding protein (TBP)-associated factor 9 allele (TAF9). Characterized core elements, including TATA box-flanking regions and the downstream promoter element, were found to play significant roles in determining promoter strength, response to activators, and factor occupancy and recruitment. The requirement for TAF9 was found to be highly promoter specific, and TAF9 dependence and promoter occupancy were not always correlated. We also describe contrasting examples of factor recruitment and activation mechanisms at different promoters, highlighted by the nearly opposite mechanisms utilized by the simian virus 40 enhancer and p53. With the core promoters analyzed, the former functions by facilitating RNA polymerase II (RNAP II) recruitment to a preassembled TBP/TFIIB-containing scaffold and p53 strongly recruits TBP and TFIIB while RNAP II levels remain modest. Taken together, our results illustrate both the important roles of core promoter elements and the remarkable diversity that characterizes transcriptional activation mechanisms in vertebrates. PMID: 14517303 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Cell Cycle. 2003 Sep-Oct;2(5):442-4. The TATA-binding protein as a regulator of cellular transformation. Johnson SA, Dubeau L, White RJ, Johnson DL. Department of Biochemistry and Molecular Biology, Keck School of Medicine and Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, California 90033, USA. The TATA-binding protein, TBP, is used by all three RNA polymerases and is therefore central to the process of gene expression. TBP associates with several subsets of proteins, called TATA-binding protein-associated factors (TAFs). This results in the formation of at least three distinct complexes, SL1, TFIID, and TFIIIB, which dictates whether TBP functions in RNA polymerase (pol) I, pol II, or pol III transcription, respectively. The regulation of gene expression has focused largely on proteins that serve to modulate the efficiency by which the general transcription components, such as TBP, interact with promoters. The possibility of a basal transcription factor, itself, being regulated, and influencing cellular homeostasis, has not been extensively considered. However, recent studies have indicated that TBP is indeed regulated, and that modulation of its cellular concentration has a profound, and surprisingly selective, impact on gene expression that can mediate the normal proliferative responses of cells to growth stimuli as well as the transformation potential of cells. Publication Types: Review Review, Tutorial PMID: 12963838 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: EMBO J. 2003 Jun 2;22(11):2810-20. p53 represses RNA polymerase III transcription by targeting TBP and inhibiting promoter occupancy by TFIIIB. Crighton D, Woiwode A, Zhang C, Mandavia N, Morton JP, Warnock LJ, Milner J, White RJ, Johnson DL. Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, Davidson Building, University of Glasgow, Glasgow G12 8QQ, UK. The tumor suppressor p53 is a transcription factor that controls cellular growth and proliferation. p53 targets include RNA polymerase (pol) III-dependent genes encoding untranslated RNAs such as tRNA and 5S rRNA. These genes are repressed through interaction of p53 with TFIIIB, a TATA-binding protein (TBP)-containing factor. Although many studies have shown that p53 binds to TBP, the significance of this interaction has remained elusive. Here we demonstrate that the TBP-p53 interaction is of functional importance for regulating RNA pol III-transcribed genes. Unlike RNA pol II-dependent promoter repression, overexpressing TBP can reverse inhibition of tRNA gene transcription by p53. p53 does not disrupt the direct interaction between the TFIIIB subunits TBP and Brf1, but prevents the association of Brf1 complexes with TFIIIC2 and RNA pol III. Using chromatin immunoprecipitation assays, we found that TFIIIB occupancy on tRNA genes markedly decreases following p53 induction, whereas binding of TFIIIC2 to these genes is unaffected. Together our results support the idea that p53 represses RNA pol III transcription through direct interactions with TBP, preventing promoter occupancy by TFIIIB. PMID: 12773395 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: J Virol. 2002 Dec;76(24):12503-12. The Epstein-Barr virus immediate-early protein BZLF1 regulates p53 function through multiple mechanisms. Mauser A, Saito S, Appella E, Anderson CW, Seaman WT, Kenney S. Department of Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA. The Epstein-Barr virus (EBV) immediate-early protein BZLF1 is a transcriptional activator that mediates the switch between the latent and the lytic forms of EBV infection. It was previously reported that BZLF1 inhibits p53 transcriptional function in reporter gene assays. Here we further examined the effects of BZLF1 on p53 function by using a BZLF1-expressing adenovirus vector (AdBZLF1). Infection of cells with the AdBZLF1 vector increased the level of cellular p53 but prevented the induction of p53-dependent cellular target genes, such as p21 and MDM2. BZLF1-expressing cells had increased p53-specific DNA binding activity in electrophoretic mobility shift assays, increased p53 phosphorylation at multiple residues (including serines 6, 9, 15, 33, 46, 315, and 392), and increased acetylation at lysine 320 and lysine 382. Thus, the inhibitory effects of BZLF1 on p53 transcriptional function cannot be explained by its effects on p53 phosphorylation, acetylation, or DNA binding activity. BZLF1 substantially reduced the level of cellular TATA binding protein (TBP) in both normal human fibroblasts and A549 cells, and the inhibitory effects of BZLF1 on p53 transcriptional function could be partially rescued by the overexpression of TBP. Thus, BZLF1 has numerous effects on p53 posttranslational modification but may inhibit p53 transcriptional function in part through an indirect mechanism involving the suppression of TBP expression. PMID: 12438576 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Biochim Biophys Acta. 2002 Oct 21;1592(2):193-204. Hepatitis C virus NS5A protein binds TBP and p53, inhibiting their DNA binding and p53 interactions with TBP and ERCC3. Qadri I, Iwahashi M, Simon F. Department of Medicine, Division of Gastroenterology and Hepatology, Hepatobiliary Center, University of Colorado Health Sciences Center, B-145, 4200 E., 9th Avenue, Denver 80262, USA. Ishtiaq.qadri@uchsc.edu Among the hepatotropic viruses, hepatitis C virus (HCV) is considered to be the leading cause of liver disease in humans, affecting approximately 2% of the world population. HCV-encoded nonstructural protein 5A (NS5A) is a 56-58-kDa phosphoprotein, which is produced from the processing of viral polyprotein. The potential mechanism(s) by which NS5A is able to influence key cellular processes are largely unknown. In this study, we investigated the functional properties of NS5A. In vivo co-immunoprecipitation and pull-down assays demonstrated that NS5A forms a heteromeric complex with TATA box binding protein (TBP) and tumor suppressor protein p53. Mutants of TBP and p53 showed reduced binding to NS5A. To determine the functional relevance of these associations, we found that NS5A inhibits the binding of both p53 and TBP to their DNA consensus binding sequences in vitro. NS5A also inhibited the p53-TBP and p53-excision repair cross complementing factor 3 (ERCC3) protein-protein complex formation. Furthermore, NS5A repressed the p53 regulated p21 (WAF1) promoter and a synthetic promoter containing multiple p53 responsive DNA elements binding sites in HCT116 p53(+/+) cell line. p53-mediated transcriptional activation from both promoters was reduced approximately 3-5-fold following expression of NS5A.Taken together, these results suggest that NS5A may exert its influence on key cellular processes by functional associations with p53 and TBP. This could explain one of the possible mechanism(s) by which NS5A is able to exert its effect on cellular gene expression and cell growth regulation. PMID: 12379483 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Exp Cell Res. 2002 Oct 1;279(2):248-59. Repression of transcription and interference with DNA binding of TATA-binding protein by C-terminal alternatively spliced p53. Huang H, Kaku S, Knights C, Park B, Clifford J, Kulesz-Martin M. Department of Experimental Therapuetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. The protein encoded by C-terminal alternatively spliced p53 mRNA (p53as) has been shown previously to occur naturally in mouse cells and to bind sequence-specifically to DNA more efficiently than p53 (p53r, regular form). In the current study, p53as and p53r proteins ectopically expressed in p53-deficient cells each transactivated reporter plasmids containing p53 binding sites. However, p53as consistently was more efficient in transcriptional repression of promoters lacking p53 binding sites and in concentration-dependent repression of the p21(WAF1/Cip-l/Sdi) promoter sequence. The p53as protein, like p53r, associated with TATA-binding protein (TBP), indicating that this interaction does not require the last 26 amino acids of p53. Consistent with its stronger repression effects, p53as interfered with TBP binding to a TATA-containing DNA sequence more efficiently than p53r protein. Taken together, these in vitro and in vivo results demonstrate a novel role in transcriptional repression for a naturally occurring C-terminal variant form of mouse p53 protein associated with differences in DNA binding properties and interference with transcription factor binding. PMID: 12243750 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: J Biol Chem. 2002 Oct 18;277(42):39769-76. Epub 2002 Aug 6. p73 independent of c-Myc represses transcription of platelet-derived growth factor beta-receptor through interaction with NF-Y. Hackzell A, Uramoto H, Izumi H, Kohno K, Funa K. Department of Cell Biology, Institute of Anatomy and Cell Biology, Goteborg University, Box 420, SE-405 30 Gothenburg, Sweden. We recently reported that c-Myc represses the transcription of platelet-derived growth factor (PDGF) beta-receptor (Izumi, H., Molander, C., Penn, L. Z., Ishisaki, A., Kohno, K., and Funa, K. (2001) J. Cell Sci. 114, 1533-1544). We demonstrate here that the p53 family protein p73alpha represses PDGF beta-receptor transcription essentially by the same mechanism. p73alpha but not p73beta or p53 represses the transcription in concordance with its ability to bind NF-YC and NF-YB. None of other p73 isoforms (i.e. p73beta, p73gamma, p73epsilon), C-terminal deletion mutants of p73alpha, and p53 is able to bind NF-Y with the exception of p63alpha. This finding suggests that the sterile alpha-motif domain present only in p73alpha and p63alpha is the interaction site. For the repression, the N-terminal transactivation domain of p73alpha is also indispensable, arguing for the importance of the activity of p73alpha in the mechanism. p73alpha binds the C-terminal HAP domain of NF-YC previously found to be the interaction site with c-Myc and TBP. Because c-Myc induces and activates p73alpha (Zaika, A., Irwin, M., Sansome, C., and Moll, U. M. (2001) J. Biol. Chem. 276, 11310-11316) and they bind each other (Uramoto, H., Izumi, H., Ise, T., Tada, M., Uchiumi, T., Kuwano, M., Yasumoto, K., Funa, K., and Kohno, K. (2002) J. Biol. Chem. 277, in press), we examined whether the repression by p73 is dependent on c-Myc. However, Myc-null rat fibroblasts are also susceptible to p73alpha-induced repression. Serum stimulation of NIH3T3 cells gradually decreased the amount of endogenous NF-Y binding to the PDGF beta-receptor promoter, whereas NF-YA expression in the nuclear extracts remains unchanged. Our results indicate that serum stimulation induces c-Myc and p73alpha, leading to the down-regulation of PDGF beta-receptor expression by repressing its transcription. PMID: 12167641 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Virology. 2001 Dec 20;291(2):260-71. Poliovirus 3C protease-mediated degradation of transcriptional activator p53 requires a cellular activity. Weidman MK, Yalamanchili P, Ng B, Tsai W, Dasgupta A. Department of Microbiology, Immunology, and Molecular Genetics, School of Medicine, University of California Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095-1747, USA. Infection of HeLa cells with poliovirus leads to rapid shut-off of host cell transcription by RNA polymerase II. Previous results have suggested that both the basal transcription factor TBP (TATA-binding protein) and transcription activator proteins such as CREB (cyclic AMP-responsive element-binding protein) and Oct-1 (the octamer-binding factor) are cleaved by the viral-encoded protease, 3C(Pro). Here we demonstrate that the transcriptional activator (and tumor suppressor) p53 is degraded by the viral protease 3C both in vivo and in vitro. Unlike other transcription factors that are directly cleaved by 3C(pro), degradation of p53 requires a HeLa cell activity in addition to 3C(Pro). The degradation of p53 by 3C(Pro) does not appear to involve the ubiquitin pathway of protein degradation. Vaccinia virus infection of HeLa cells leads to inactivation of the cellular activity required for 3C(Pro)-mediated degradation of p53. The vaccinia-encoded protein (CrmA) is known to inhibit caspase I (ICE protease) that converts inactive IL-1beta to an active secreted form. Incubation of HeLa cells with caspase I inhibitor Z-VAD-fmk does not interfere with 3C(Pro)-mediated degradation of p53. The cellular activity present in extracts of HeLa cells can be fractionated through phosphocellulose. A partially purified fraction that elutes at 0.6 M KCl from phosphocellulose contains the activity that degrades p53 in a 3C(Pro)-dependent manner. These results suggest that both poliovirus-encoded protease 3C(Pro) and a cellular activity are required for the degradation of p53 observed in cells infected with poliovirus. (C)2001 Elsevier Science PMID: 11878895 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Oncogene. 2001 Jan 11;20(2):240-51. Negative regulation of bcl-2 expression by p53 in hematopoietic cells. Wu Y, Mehew JW, Heckman CA, Arcinas M, Boxer LM. The Center for Molecular Biology in Medicine, Palo Alto Veterans Affairs Medical Center and the Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305-5112, USA. The p53 protein activates promoters containing p53 binding sites, and it represses other promoters. We examined the effect of p53 on bcl-2 expression in both the DHL-4 B cell line and the K562 erythroleukemia line. Transient transfection analyses revealed that wild-type p53 repressed the bcl-2 full-length promoter. The region of the bcl-2 promoter that was responsive to p53 was mapped to the bcl-2 P2 minimal promoter region, and we showed that p53 and the TATA binding protein bound to the bcl-2 TATA sequence. The TATA binding protein, p53, histone deacetylase-1 and mSin3a could be co-immunoprecipitated from K562 cell nuclear extract. The TATA binding protein and mSin3a could be recovered in a complex at the bcl-2 promoter TATA sequence, however, the formation of this complex was not dependent on the presence of p53. Treatment of K562 cells with the histone deacetylase inhibitor, trichostatin A, resulted in an increase in bcl-2 promoter activity whether p53 was present or not. Therefore, we demonstrated that p53 and the histone deacetylases repress the bcl-2 promoter independently. Similar results were obtained when endogenous bcl-2 mRNA or protein levels were measured in response to either p53 or trichostatin A, and p53 expression resulted in enhanced apoptosis. RNase protection assays demonstrated that transcription from the endogenous 3' bcl-2 promoter was decreased by p53. The regions of p53 that were required for repression of the bcl-2 promoter were defined. We conclude that the TATA sequence in the bcl-2 P2 minimal promoter is the target for repression by p53, and that the interaction between p53 and TBP is most likely responsible for the repression. Mutation of p53 may play a role in the up-regulation of bcl-2 expression in some B cell lymphomas. PMID: 11313951 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Oncogene. 2000 Aug 3;19(33):3717-26. Activation of the insulin-like growth factor II transcription by aflatoxin B1 induced p53 mutant 249 is caused by activation of transcription complexes; implications for a gain-of-function during the formation of hepatocellular carcinoma. Lee YI, Lee S, Das GC, Park US, Park SM, Lee YI. Bioscience Research Division, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon. Aflatoxin B1 (AFB1) induced mutation of the p53 gene at codon 249 (p53mt249) is critical during the formation of hepatocellular carcinoma (HCC) following hepatitis B virus (HBV) infection. p53mt249 markedly increases insulin-like growth factor II (IGF-II) transcription largely from promoter 4, accumulating the fetal form of IGF-II. Modulation of the transcription factor binding to IGF-II P4 by wild-type p53 and p53mt249 was identified. Wild-type p53 inhibited binding of transcription factors Sp1 and TBP on the P4 promoter, while p53mt249 enhanced the formation of transcriptional complexes through enhanced DNA-protein (Sp1 or TBP) and protein-protein (Sp1 and TBP) interactions. p53mt249 stimulates transcription factor Sp1 phosphorylation which might be a cause of increased transcription factor binding on the P4 promoter while wild-type p53 does not. Transfection of hepatocytes with p53mt249 impaired induction of apoptosis by the HBV-X protein and TNF-alpha. Therefore, the blocking of apoptosis through enhanced production of IGF-II should provide a favorable opportunity for the selection of transformed hepatocytes. These results explain the molecular basis for the genesis of HCC by p53mt249 which was found to be induced by a potent mutagen, AFB1. PMID: 10949925 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: EMBO J. 1998 Jun 1;17(11):3112-23. p53 is a general repressor of RNA polymerase III transcription. Cairns CA, White RJ. Institute of Biomedical and Life Sciences, Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow, G12 8QQ, UK. p53 is a major tumour suppressor that is inactivated in a large proportion of human cancers. We show that p53 serves as a general repressor of transcription by RNA polymerase (pol) III. It can inhibit the synthesis of a range of essential small cellular RNAs including tRNA, 5S rRNA and U6 snRNA, as well as viral products such as the adenovirus VAI RNA. Fibroblasts derived from p53 knock-out mice display a substantial increase in pol III transcriptional activity. Endogenous cellular p53 is shown to interact with the TATA-binding protein (TBP)-containing general factor TFIIIB, thereby compromising its function severely. However, assembly of TFIIIB into a pre-initiation complex confers substantial protection against the inhibitory effects of p53. Since TFIIIB is an essential determinant of the biosynthetic capacity of cells, its release from repression by p53 may contribute to a loss of growth control during the development of many tumours. PMID: 9606193 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: DNA Cell Biol. 1998 Feb;17(2):125-31. p53 regulates human insulin-like growth factor II gene expression through active P4 promoter in rhabdomyosarcoma cells. Zhang L, Zhan Q, Zhan S, Kashanchi F, Fornace AJ Jr, Seth P, Helman LJ. Molecular Oncology Section, Pediatric Oncology Branch, National Cancer Institute, Bethesda, MD 20892-1928, USA. The developmentally regulated human insulin-like growth factor II (IGFII) gene is expressed at high levels in many types of tumors and promotes the proliferation of tumor cells with a high incidence of p53 gene defects. We have previously shown that p53 inhibits IGFII P3 promoter activity and decreases endogenous IGFII gene expression derived from the P3 promoter in rhabdomyosarcomas by interfering with TBP binding to the TATA element of the IGFII P3 promoter. In this report, we demonstrate that wild-type p53 expression in rhabdomyosarcoma cell lines containing mutant p53 leads to a decrease in the activity of another active IGFII promoter, P4, and a 5-fold reduction of IGFII mRNA derived from the P4 promoter. This inhibition of P4 activity is associated with direct binding of p53 to the P4 proximal promoter element despite the lack of a p53 consensus binding site. Our results suggest that p53 inhibits IGFII P4 promoter activity by a mechanism different than its effect on the P3 promoter. These data also supply further evidence of cross-talk between the IGF and p53 signaling pathways. PMID: 9502429 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: J Gen Virol. 1997 Oct;78 ( Pt 10):2607-13. Human papillomavirus type 16 E7 binds to the conserved carboxy-terminal region of the TATA box binding protein and this contributes to E7 transforming activity. Massimi P, Pim D, Banks L. International Centre for Genetic Engineering and Biotechnology, Trieste, Italy. We have previously shown that the human papillomavirus E7 proteins bind to the cellular TATA box binding protein (TBP). In this paper we show that the HPV-18 E6 and the HPV-16 E2 proteins will also bind TBP in vitro. This feature of virus proteins is conserved across many viral types and we were interested in determining whether these HPV proteins interacted with the same conserved region of the TBP molecule. A series of deletions was introduced into the TBP protein and its binding to these HPV proteins was measured. The previously well-characterized interaction between p53 and TBP was used for comparison. All four proteins were found to interact with the carboxy-terminal domain of the TBP protein, although the precise residues involved and the relative strengths of association differed between the different HPV proteins. Mutational analysis of HPV-16 E7 protein identified a stretch of four amino acids responsible for the binding to TBP. This mutant E7 protein possessed wild-type levels of transcriptional activity on the adenovirus E2 promoter but exhibited reduced transforming activity in cooperation with EJ-ras. These results demonstrate that the mechanisms of interaction between diverse viral proteins and TBP are similar and that, in the case of E7, this interaction may contribute to its transforming activity. PMID: 9349482 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Blood. 1997 Dec 15;90(12):4924-32. Repression of transcription from the human T-cell leukemia virus type I long terminal repeat and cellular gene promoters by wild-type p53. Mori N, Kashanchi F, Prager D. Division of Hematology/Oncology, University of California at Los Angeles School of Medicine, Los Angeles, CA, USA. Human T-cell leukemia virus type-I (HTLV-I), the etiologic agent of adult T-cell leukemia (ATL) transforms human T cells both in vivo and in vitro. However, the long latency period between infection and development of ATL, as well as the small fraction of the infected population that actually develops this disease, suggest that factors in addition to the virus are involved in its pathogenesis. Mutation of tumor suppressor gene p53 has been found in both HTLV-I-transformed T-cell lines and ATL cases at relatively low frequency. However, increasing evidence supports p53 functional impairment in HTLV-I-transformed T cells. Tax, the major transactivator of HTLV-I, is critical for the initial events involved in transformation. We have considered the possibility that p53 may regulate transcription of viral and cellular genes important for viral replication and transformation. Inactivation of p53 function might then permit constitutive expression of these viral and cellular genes. We have investigated the effects of wild-type and mutant p53 on Tax-mediated activation of the HTLV-I long terminal repeat (LTR) and the promoters of several cellular genes including the interleukin (IL)-1alpha, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF ), and IL-2 receptor alpha chain gene. Jurkat, HuT78, and U937 cells were cotransfected with plasmids containing a chloramphenicol acetyltransferase (CAT ) reporter gene under viral or cellular promoter control and the Tax expression vector, in addition to vectors for a wild-type or mutant p53. Wild-type p53 is a potent repressor of viral and cellular activation by Tax. Mutations within p53 severely inhibit this downregulation. We also show that wild-type p53 suppresses transcription from the HTLV-I LTR in Jurkat-Tax, a T-cell line stably expressing Tax, and MT-2, a HTLV-I-transformed T-cell line. Wild-type, but not mutant, p53 interfered with the binding of TATA-binding protein (TBP) to the TATA motif of the HTLV-I LTR. These results suggest that p53 inactivation may lead to upregulation of viral and cellular genes and may also be important for establishment of productive viral infection and development of ATL. PMID: 9389710 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Oncogene. 1997 Oct;15(18):2191-9. A functional analysis of p53 during early development of Xenopus laevis. Amariglio F, Tchang F, Prioleau MN, Soussi T, Cibert C, Mechali M. Institut Jacques Monod, CNRS, Paris, France. p53 is a nuclear protein that acts like a tumor suppressor and is involved in regulation of cellular growth. In Xenopus, the p53 protein is highly expressed during oogenesis and is strictly cytoplasmic in the oocyte. We have analysed its participation in DNA replication and transcription during early development, using the egg and oocyte as model-systems. The injection of sperm nuclei into Xenopus eggs is followed by DNA replication and mitotic events. We show that the endogenous p53 enters the nuclei and moves through a series of discrete sub-nuclear loci whose distribution is S-phase specific. A specific peripheral nuclear localization of p53 is observed before entry into S-phase, followed by an internal localization which is strictly dependent on ongoing DNA synthesis. At no stage in the cell cycle, however, did we observe any co-localization with RPA or PCNA, which were used as initiation or elongation markers for DNA replication. We also show that injection into the nucleus of the oocyte of small amounts of either Xenopus or human p53 - less than 10% of the cytoplasmic storage - is sufficient to block RNA polymerase II-dependent transcription from a coinjected TATA-box-containing reporter plasmid. Transcription is rescued by microinjection of the TATA-box binding protein (TBP), suggesting that nuclear exclusion of p53 during oogenesis may be necessary for transcription of maternal genes. These characteristics are discussed in relation to the regulation of nuclear activities during early embryogenesis. PMID: 9393977 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: J Cell Biochem. 1997 Sep 1;66(3):277-85. Roles of p300, pocket proteins, and hTBP in E1A-mediated transcriptional regulation and inhibition of p53 transactivation activity. Sang N, Avantaggiati ML, Giordano A. Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. The conserved region 1 and the extreme N-terminus of adenoviral oncoprotein E1A are essential for transforming activity. They also play roles in the interaction of E1A with p300/CBP and pRb and are involved in both transactivation and repression of host gene expression. It was reported recently that p53-mediated transactivation is specifically repressed by E1A and that p53-induced apoptosis can be protected by pRb. In this report, we investigated the roles of pRb and p300 in the N-terminus of E1A-mediated transcriptional regulation. We demonstrate here that p300 and pRb have no effect on DBD.1-70 transactivation and that overexpression of p300 or pRb failed to relieve the repression by E1A. Repression of p53 transactivation requires both the extreme amino terminus and CR1 but not CR2. This repressive activity of E1A specifically correlates with E1A's ability to bind p300 and TBP. On the other hand, E1A inhibited the transactivation activity of a fusion construct containing the DNA binding domain of yeast Gal4 and the transactivation domain of p53. When p53 was contransfected with E1A, similar inhibition was found in Saos-2 cells that lack endogenous pRb and p53 activity. Introduction of pRb into Saos-2 cells did not affect p53 transcription activity. E1A-mediated repression can be relieved be overexpression of either p300, hTBP, or-TFIIB but cannot be released by overexpression of pocket proteins. Our data suggest that p300/CBP and TBP but not the pocket proteins, pRb, p107, and pRb2/p130 are functional targets of E1A in transcriptional regulation and that p53 transactivation requires the function of the p300/TBP/TFIIB complex, thus delineating a new pathway by which E1A may exert its transforming activity. PMID: 9257185 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Genes Dev. 1997 Aug 1;11(15):1974-86. Repression of p53-mediated transcription by MDM2: a dual mechanism. Thut CJ, Goodrich JA, Tjian R. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720-3204, USA. The oncoprotein MDM2 binds to the activation domain of the tumor suppressor p53 and inhibits its ability to stimulate transcription. This same region of p53 is able to bind several basal transcription factors that appear to be important for the transactivation function of p53. It has therefore been suggested that MDM2 acts to inhibit p53 by concealing its activation domain from the basal machinery. Here we present data suggesting that MDM2 possesses an additional inhibitory function. Our experiments reveal that in addition to a p53-binding domain, MDM2 also contains an inhibitory domain that can directly repress basal transcription in the absence of p53. By fusing portions of MDM2 to a heterologous DNA-binding domain to allow p53-independent promoter recruitment, we have localized this inhibitory domain to a region encompassing amino acids 50-222 of MDM2. Furthermore, the function of this inhibitory domain does not require the presence of either TFIIA or the TAFs. Of the remaining basal factors, both the small subunit of TFIIE and monomeric TBP are bound by the MDM2 inhibitory domain. It is possible that MDM2 inhibits the ability of the preinitiation complex to synthesize RNA through one of these interactions. Our results are consistent with a model in which MDM2 represses p53-dependent transcription by a dual mechanism: a masking of the activation domain of p53 through a protein-protein interaction that additionally serves to recruit MDM2 to the promoter where it directly interferes with the basal transcription machinery. PMID: 9271120 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Virology. 1997 Jan 6;227(1):255-9. Repression of p53 transcriptional activity by the HPV E7 proteins. Massimi P, Banks L. International Centre for Genetic Engineering and Biotechnology, Padriciano 99, Trieste, I-34012, Italy. The major transforming protein of human papillomaviruses (HPVs) is encoded by the E7 gene. This protein cooperates with activated oncogenes to transform primary rodent cells and with the viral E6 gene to immortalize primary human keratinocytes. Numerous cellular targets of HPV E7 have now been identified including pRb, p107, cyclin A, TATA box binding protein (TBP), and members of the AP-1 transcription factor family. As with Adenovirus E1a, many of these interactions are important for the ability of E7 to transform cells. Recent studies have demonstrated that Adenovirus E1a can also inhibit the transcriptional activity of the cellular tumor suppressor protein, p53. We have performed a series of analyses to determine whether HPV E7 proteins share this characteristic. We show that HPV E7 proteins derived from both benign and tumor-associated HPV types are able to inhibit p53 transcriptional activity. Mutational analysis of the HPV-16 E7 protein reveals that a key domain involved in mediating this activity is the casein kinase II (CKII) recognition site, which has been shown to modulate E7 binding to TBP. We further show that E7 does not bind to p53 directly, but will do so in the presence of exogenously added TBP and that this binding is increased following CKII phosphorylation. These results suggest that the E7-TBP interaction may be responsible for inhibiting p53 transcriptional activity. PMID: 9007083 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Mol Cell Biol. 1996 Dec;16(12):7084-8. p53 inhibits RNA polymerase III-directed transcription in a promoter-dependent manner. Chesnokov I, Chu WM, Botchan MR, Schmid CW. Section of Molecular and Cellular Biology, University of California, Davis 95616, USA. Wild-type p53 represses Alu template activity in vitro and in vivo. However, upstream activating sequence elements from both the 7SL RNA gene and an Alu source gene relieve p53-mediated repression. p53 also represses the template activity of the U6 RNA gene both in vitro and in vivo but has no effect on in vitro transcription of genes encoding 5S RNA, 7SL RNA, adenovirus VAI RNA, and tRNA. The N-terminal activation domain of p53, which binds TATA-binding protein (TBP), is sufficient for repressing Alu transcription in vitro, and mutation of positions 22 and 23 in this region impairs p53-mediated repression of an Alu template both in vitro and in vivo. p53's N-terminal domain binds TFIIIB, presumably through its known interaction with TBP, and mutation of positions 22 and 23 interferes with TFIIIB binding. These results extend p53's transcriptional role to RNA polymerase III-directed templates and identify an additional level of Alu transcriptional regulation. PMID: 8943363 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Nucleic Acids Res. 1996 Nov 1;24(21):4281-8. Transcriptional repression by p53 involves molecular interactions distinct from those with the TATA box binding protein. Farmer G, Friedlander P, Colgan J, Manley JL, Prives C. Department of Biological Sciences, Columbia University, New York, NY 10027, USA. In addition to serving a role as a DNA binding-dependent transcriptional activator, p53 has been reported to repress a variety of promoters that lack p53 binding sites. Data from recent studies have suggested that this activity is mediated via an interaction between p53 and the TATA box binding protein (TBP). To investigate the functional relevance of this interaction in vivo, we have performed transient transfection assays in Drosophila Schneider cells. Wild-type p53 was found to repress expression from TATA box- but not initiator (Inr)-containing promoters activated by GAL4-VP16, GAL4-ftzQ or Sp1. A mutant p53(His175), defective in DNA binding and transcriptional activation, also inhibited TATA-dependent transcription activated by Sp1. However, p53 was unable to repress a basal TATA promoter stimulated by overexpression of TBP. Furthermore, overexpression of TBP failed to rescue the p53-mediated repression of activated transcription and a p53 mutant with its N-terminal TBP interaction domain intact, but defective in transcriptional activation and binding to TBP-associated factors (TAFs), was similarly defective in transcriptional repression. These data suggest that a p53-TBP interaction is not sufficient for transcriptional repression by p53 and that repression involves an interaction between p53 and other factors, such as TAFs, that are required for activated but not basal transcription. We suggest that p53-mediated repression results from squelching of a factor limiting for activated transcription from TATA- but not Inr-containing promoters. PMID: 8932384 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8318-23. Wild-type and mutant p53 differentially regulate transcription of the insulin-like growth factor I receptor gene. Werner H, Karnieli E, Rauscher FJ, LeRoith D. Section on Molecular and Cellular Physiology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. The insulin-like growth factor I receptor (IGF-I-R) plays a critical role in transformation events. It is highly overexpressed in most malignant tissues where it functions as an anti-apoptotic agent by enhancing cell survival. Tumor suppressor p53 is a nuclear transcription factor that blocks cell cycle progression and induces apoptosis. p53 is the most frequently mutated gene in human cancer. Cotransfection of Saos-2 (os-teosarcoma-derived cells) and RD (rhabdomyosarcoma-derived cells) cells with IGF-I-R promoter constructs driving luciferase reporter genes and with wild-type p53 expression vectors suppressed promoter activity in a dose-dependent manner. This effect of p53 is mediated at the level of transcription and it involves interaction with TBP, the TATA box-binding component of TFIID. On the other hand, three tumor-derived mutant forms of p53 (mut 143, mut 248, and mut 273) stimulated the activity of the IGF-I-R promoter and increased the levels of IGF-I-R/luciferase fusion mRNA. These results suggest that wild-type p53 has the potential to suppress the IGF-I-R promoter in the postmitotic, fully differentiated cell, thus resulting in low levels of receptor gene expression in adult tissues. Mutant versions of p53 protein, usually associated with malignant states, can derepress the IGF-I-R promoter, with ensuing mitogenic activation by locally produced or circulating IGFs. PMID: 8710868 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: J Interferon Cytokine Res. 1996 Aug;16(8):595-600. Repression of interleukin-2 and interleukin-4 promoters by tumor suppressor protein p53. Pesch J, Brehm U, Staib C, Grummt F. Institut fur Biochemie, Universitat Wurzburg, Germany. Interleukin 2 (IL-2) and interleukin 4 (IL-4) secreted by activated but not by resting mature T cells are pleiotropic cytokines affecting growth and differentiation of diverse cell types, such as T cells, B cells, and mast cells. There is little information about the molecular basis for the constitutive repression of IL-2 and IL-4 gene expression in unstimulated T cells. We investigated the possibility that wild-type (wt) p53, a nuclear tumor suppressor protein, might serve to repress IL-2 and IL-4 gene expression in murine E14 T lymphoma and in human Jurkat cells. We transiently cotransfected these cells with constitutive simian virus 40 (SV 40) early promoter expression plasmids overproducing wt or mutant murine p53 and with appropriate luciferase (luc) reporter plasmids containing the promoter elements of murine IL-2 and IL-4 genes to evaluate the effect of various p53 species on these promoters. Murine wt p53 derived from pSG5p53cD strongly repressed the IL-2 and IL-4 promoters in both cell lines induced by the phorbol ester TPA and the Ca2+ ionophore ionomycin but not, however, in uninduced cells. In similar transient transfection experiments with lymphoma cells, overexpression of deletion mutant species of murine p53 revealed that the N-terminal and C-terminal domains are crucial for inhibition of both IL-2 and IL-4 gene expression. These parts of p53 comprise the transactivation domain at the amino terminal side, which has previously also been shown to interact with the TATA-box binding-protein TBP and the carboxy-terminal oligomerization domain. Additionally, it was shown that a previously described inhibitory protein, the high-mobility-group protein HMG-I/Y, does not functionally interact with p53. Cotransfection of expression plasmids for both p53 and HMG-I/Y did not alter the extent of inhibition by the individual proteins. These data suggest that p53 can downmodulate both IL-2 and IL-4 gene expression and that both the transactivation and oligomerization domains of the tumor suppressor protein are essential for this transcriptional repression. PMID: 8877730 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Oncogene. 1996 Aug 1;13(3):471-80. HPV-18 E6 inhibits p53 DNA binding activity regardless of the oligomeric state of p53 or the exact p53 recognition sequence. Thomas M, Massimi P, Banks L. International Centre for Genetic Engineering and Biotechnology, Trieste, Italy. The E6 proteins of the oncogenic-associated human papillomavirus types 16 (HPV-16) and 18 (HPV-18) function by interfering with the normal cell cycle control mechanisms, particularly those controlled by p53. HPV E6 is able to interfere with p53 function by preventing its binding to DNA target sequences and also by labelling p53 for ubiquitin-mediated degradation. We have previously reported that certain p53 mutants, defective in oligomerisation, vary in their susceptibility to E6-directed labelling for ubiquitin-mediated degradation. In this paper we report that the strength of p53's binding to DNA is dependent upon the precise target sequence, but that E6 is able to disrupt each complex. We also report the binding of different oligomeric forms of p53 to different DNA sequences and correlate this with in vivo transcriptional activity and demonstrate the susceptibility of that DNA binding to disruption by E6. Finally we show that the ability of p53 to bind to TBP is a function of its oligomeric state and correlates in part with its ability to transrepress but not with its ability to transactivate. PMID: 8760288 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Mol Cell Biol. 1996 Aug;16(8):4295-304. Functional interaction between p53, the TATA-binding protein (TBP), andTBP-associated factors in vivo. Farmer G, Colgan J, Nakatani Y, Manley JL, Prives C. Department of Biological Sciences, Columbia University, New York, New York 10027, USA. The transcriptional activator p53 is known to interact with components of the general transcription factor TFIID in vitro. To examine the relevance of these associations to transcriptional activation in vivo, plasmids expressing a p53-GAL4 chimera and Drosophila TATA-binding protein (dTBP) were transfected into Drosophila Schneider cells. p53-GAL4 and dTBP displayed a markedly synergistic effect on activated transcription from a GAL4 site-containing reporter that was at least 10-fold greater than observed with other activators tested. A mutant p53 previously shown to be defective in both transcriptional activation in vivo and in binding to TBP-associated factors (TAFs) in vitro, although still capable of binding dTBP, did not cooperate with dTBP, suggesting that TAFs may contribute to this synergy. Providing further support for this possibility, transfected dTBP assembled into rapidly sedimenting complexes and could be immunoprecipitated with anti-TAF antibodies. While overexpression of any of several TAFs did not affect basal transcription, in either the presence or the absence of cotransfected dTBP, overexpression of TAFII230 inhibited transcriptional activation mediated by p53-GAL4 as well as by GAL4-VP16 and Sp1. Overexpression of TAFII40 and TAFII60 also inhibited activation by p53-GAL4 but had negligible effects on activation by GAL4-VP16 and Sp1, while TAFII110 did not affect any of the activators. TAF-mediated inhibition of activated transcription could be rescued by high levels of exogenous dTBP, which also restored full synergy. These data demonstrate for the first time that functional interactions can occur in vivo between TBP, TAFs, and p53. PMID: 8754830 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10550-4. The ability to associate with activation domains in vitro is not required for the TATA box-binding protein to support activated transcription in vivo. Tansey WP, Herr W. Cold Spring Harbor Laboratory, NY 11724, USA. The TATA box-binding protein (TBP) interacts in vitro with the activation domains of many viral and cellular transcription factors and has been proposed to be a direct target for transcriptional activators. We have examined the functional relevance of activator-TBP association in vitro to transcriptional activation in vivo. We show that alanine substitution mutations in a single loop of TBP can disrupt its association in vitro with the activation domains of the herpes simplex virus activator VP16 and of the human tumor suppressor protein p53; these mutations do not, however, disrupt the transcriptional response of TBP to either activation domain in vivo. Moreover, we show that a region of VP16 distinct from its activation domain can also tightly associate with TBP in vitro, but fails to activate transcription in vivo. These data suggest that the ability of TBP to interact with activation domains in vitro is not directly relevant to its ability to support activated transcription in vivo. PMID: 7479838 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: Mol Cell Biol. 1995 Nov;15(11):6474-8. Reversal of in vitro p53 squelching by both TFIIB and TFIID. Liu X, Berk AJ. Molecular Biology Institute, University of California, Los Angeles 90095-1570, USA. p53, the protein encoded by one of the most significant human tumor suppressor genes, is a sequence-specific transcriptional activator. When activated by a double-stranded DNA break, p53 function arrests cells in G1 and can induce apoptosis. Transcriptional activation function is critical for p53 tumor suppression, although transcriptional repressing and nontranscriptional functions of p53 may contribute. p53 activation requires that it bind to TFIID through interactions with TATA box-binding protein (TBP)-associated factors and potentially with TBP. Here, we studied the mechanism of p53 activation using in vitro transcription and a sufficiently high p53 concentration to squelch activated transcription. Squelching is thought to result when target molecules that interact with activation domains are titrated by binding to excess activator. Addition of either excess TFIIB or TFIID but not other proteins required for p53-activated transcription reversed squelching by high p53 concentrations, whereas neither stimulated transcription in reactions without excess p53. These results reveal that both TFIIB and TFIID are inhibited by high concentrations of p53 and suggest that p53 activation may work through direct or indirect interactions with both TFIIB and TFIID. PMID: 7565799 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: J Biol Chem. 1995 Oct 20;270(42):25014-9. Transactivation ability of p53 transcriptional activation domain is directly related to the binding affinity to TATA-binding protein. Chang J, Kim DH, Lee SW, Choi KY, Sung YC. Department of Life Science, Pohang University of Science and Technology, Republic of Korea. Tumor suppressor protein p53 is a potent transcriptional activator and regulates cell growth negatively. To characterize the transcriptional activation domain (TAD) of p53, various point mutants were constructed in the context of Gal4 DNA binding domain and tested for their transactivation ability. Our results demonstrated that the positionally conserved hydrophobic residues shared with herpes simplex virus VP16 and other transactivators are essential for transactivation. Also, the negatively charged residues and proline residues are necessary for full activity, but not essential for the activity of p53 TAD. Deletion analyses showed that p53 TAD can be divided into two subdomains, amino acids 1-40 and 43-73. An in vitro glutathione S-transferase pull-down assay establishes a linear correlation between p53 TAD-mediated transactivation in vivo and the binding activity of p53 TAD to TATA-binding protein (TBP) in vitro. Mutations that diminish the transactivation ability of Gal4-p53 TAD also impair the binding activity to TBP severely. Our results suggest that at least TBP is a direct target for p53 TAD and that the binding strength of TAD to TBP (TFIID) is an important parameter controlling activity of p53 TAD. In addition, circular dichroism spectroscopy has shown that p53 TAD peptide lacks any regular secondary structure in solution and that there is no significant difference between the spectra of the wild type TAD and that of the transactivation deficient mutant type. PMID: 7559631 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: Oncogene. 1995 Oct 5;11(7):1299-307. p53 represses SV40 transcription by preventing formation of transcription complexes. Perrem K, Rayner J, Voss T, Sturzbecher H, Jackson P, Braithwaite A. Division of Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia. There is now much evidence to suggest that the p53 tumour suppressor protein functions to monitor the integrity of the genome. When DNA damage is detected, p53 suppresses cell growth to allow repair or directs the cell into apoptosis. The mechanism of action of p53 is as yet unclear but recent evidence has accumulated to suggest that p53 might act by regulating gene expression. Consistent with this model, p53 can both activate and repress a number of viral and cellular promoters. p53 has also been shown to bind to the CCAAT-binding Factor and TATA-binding protein (TBP), and there is direct evidence that p53 represses in vitro transcription by preventing TBP from binding DNA. We now provide evidence that p53 can repress transcription from the SV40 promoter by disrupting DNA/protein complexes involving transcription factor Sp1. PMID: 7478550 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Oncogene. 1995 Feb 2;10(3):449-55. Cell-specific modulation of the papovavirus promoters by tumor-suppressor protein p53 in the absence of large T-antigen. Das GC, Shivakumar CV, Todd SD. Department of Molecular Biology, University of Texas Health Science Center at Tyler, 75710. The oncoproteins from several DNA tumor viruses form a complex with p53 and inactivate its function. Wild-type p53 is a transcription factor and can regulate eukaryotic promoters both positively and negatively. To elucidate the basis of the opposing functions and to understand whether and how oncoprotein synthesis in papovaviruses is regulated by p53, we studied modulation of the early promoters of SV40, polyomavirus and BK virus in the absence of the interfering effect of viral large T antigens. We here show that murine p53 can regulate the viral promoters either positively or negatively depending on the cell type. A temperature-sensitive mutant p53, 135 Val, at 37 degrees C also showed a cell-specific effect. These results suggest that promoter activation by p53 is not solely determined by p53 binding site, but host factors modulate p53's transactivation function. A TATA-less polyomavirus late promoter was also repressed in HeLa cells and the level of repression was much less in the presence of active early promoter. As p53 and 135 Val were overexpressed to similar extent in different transfected cell lines, variation in transactivation function is not due to the difference in the level of expressed protein. Our observations thus suggest that p53 interactions with cellular factors in addition to the TATA binding protein (TBP) are important for activator and repressor functions of p53. Well-defined viral promoters could thus provide us with an important tool for the identification and characterization of cellular factors that modulate p53-binding dependent gene regulation in animal cells. PMID: 7845669 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Science. 1995 Jan 6;267(5194):100-4. p53 transcriptional activation mediated by coactivators TAFII40 and TAFII60. Thut CJ, Chen JL, Klemm R, Tjian R. Howard Hughes Medical Institute, University of California, Berkeley 94720. The tumor suppressor protein p53 is a transcriptional regulator that enhances the expression of proteins that control cellular proliferation. The multisubunit transcription factor IID (TFIID) is thought to be a primary target for site-specific activators of transcription. Here, a direct interaction between the activation domain of p53 and two subunits of the TFIID complex, TAFII40 and TAFII60, is reported. A double point mutation in the activation domain of p53 impaired the ability of this domain to activate transcription and, simultaneously, its ability to interact with both TAFII40 and TAFII60. Furthermore, a partial TFIID complex containing Drosophila TATA binding protein (dTBP), human TAFII250, dTAFII60, and dTAFII40 supported activation by a Gal4-p53 fusion protein in vitro, whereas TBP or a subcomplex lacking TAFII40 and TAFII60 did not. Together, these results suggest that TAFII40 and TAFII60 are important targets for transmitting activation signals between p53 and the initiation complex. PMID: 7809597 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Science. 1994 May 27;264(5163):1326-9. Comment in: Science. 1994 May 27;264(5163):1251. The TATA-binding protein: a general transcription factor in eukaryotes and archaebacteria. Rowlands T, Baumann P, Jackson SP. Wellcome/CRC Institute, Cambridge, UK. The TATA-binding protein TBP appears to be essential for all transcription in eukaryotic cell nuclei, which suggests that its function was established early in evolution. Archaebacteria constitute a kingdom of organisms distinct from eukaryotes and eubacteria. Archaebacterial gene regulatory sequences often map to TATA box-like motifs. Here it is shown that the archaebacterium Pyrococcus woesei expresses a protein with structural and functional similarity to eukaryotic TBP molecules. This suggests that TBP's role in transcription was established before the archaebacterial and eukaryotic lineages diverged and that the transcription systems of archaebacteria and eukaryotes are fundamentally homologous. PMID: 8191287 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: Oncogene. 1994 May;9(5):1341-9. Wild-type human p53 activates the human epidermal growth factor receptor promoter. Deb SP, Munoz RM, Brown DR, Subler MA, Deb S. Department of Microbiology, University of Texas Health Science Center at San Antonio 78284. We show that wild-type human p53 transactivates the human epidermal growth factor receptor (EGFR) promoter in vivo in a dose-dependent manner, implicating p53 in promotion of cell proliferation. This activation is sensitive to the expression of cellular oncoprotein MDM2 and human papillomavirus type 18 (HPV-18) E6 protein. The p53 response element is localized within -15 and -569 of the promoter. The EGFR promoter does not have a TATA box, and has low activity in Saos-2 cells in the absence of p53. Results from our in vivo transient transfection assays suggest that p53-binding sites, without any other known promoter element, can act as bidirectional promoters in the presence of wild-type p53. Gel retardation analyses suggest that p53 may serve to nucleate TBP on a promoter. We propose that p53 successfully nucleates the transcription complex, possibly via direct interaction with TFIID, and activates the EGFR promoter. PMID: 8152794 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: Genes Dev. 1993 Oct;7(10):1837-49. Erratum in: Genes Dev 1993 Dec;7(12B):2652. Cooperative DNA binding of p53 with TFIID (TBP): a possible mechanism for transcriptional activation. Chen X, Farmer G, Zhu H, Prywes R, Prives C. Department of Biological Sciences, Columbia University, New York, New York 10027. The p53 tumor-suppressor gene product, a sequence-specific DNA-binding protein, has been shown to act both as a transcriptional activator and repressor in vivo and in vitro. Consistent with its roles in regulating transcription are recent observations that p53 binds directly to the TATA box-binding protein (TBP) subunit of the basal transcription factor TFIID. Here, we show that p53 cooperates with either recombinant TBP or partially purified TFIID in binding to a DNA fragment containing both a specific p53-binding site (RGC) and a TATA box (RGC-TATA). Surprisingly, both TBP and TFIID also stimulate p53 binding to DNA containing a specific p53-binding site but lacking a TATA box. These data are supported by the observation that p53 and Drosophila TBP combinatorily activate transcription in vivo. Our results suggest that p53 activates transcription through the formation of a more stable p53-TFIID-promoter complex. We also examined whether p53 might affect the ability of TBP or TFIID to interact with DNA containing a TATA box but lacking a p53-binding site. Although p53 strongly inhibited the interaction of TBP with such DNA, it had virtually no effect on TFIID binding. Thus, transcriptional repression by p53 may require additional functions other than inhibiting TBP binding. PMID: 8405994 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: Biochem Biophys Res Commun. 1993 Aug 31;195(1):428-34. p53 and SV40 T antigen bind to the same region overlapping the conserved domain of the TATA-binding protein. Martin DW, Subler MA, Munoz RM, Brown DR, Deb SP, Deb S. Department of Microbiology, University of Texas Health Science Center, San Antonio 78284. In this report we demonstrate that the cloned human TATA-binding protein (TBP) interacts with T antigen. TBP co-immunoprecipitates with T antigen when incubated with the T antigen-specific monoclonal antibody PAb419, and Protein-A agarose. Gel retention analysis with a radiolabeled TATA box-containing probe showed that the complex of TBP and T antigen can bind to the TATA box. Recently, p53 has also been shown to interact with TBP. Using TBP deletion mutants and co-immunoprecipitation experiments with p53 or T antigen, we show that both p53 and T antigen bind to the same region, amino acids 203-275, within the conserved C-terminal domain of TBP. Binding of p53 and T antigen to the same domain on TBP may lead to competition between the two proteins for transcriptional function. PMID: 8395834 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: J Biol Chem. 1993 Jun 25;268(18):13062-7. p53 binds to the TATA-binding protein-TATA complex. Martin DW, Munoz RM, Subler MA, Deb S. Department of Microbiology, University of Texas Health Science Center, San Antonio 78284. Earlier reports show that p53, both wild type and mutants, may affect transcription. Wild-type p53 activates promoters with p53-binding sites while inhibiting promoters without binding sites. Mutant p53, on the other hand, has been shown to activate transcription from specific promoters. These observations suggest that both wild-type and mutant p53 may interact with a general transcription factor(s). In this report, we have shown that the cloned TATA-binding protein (TBP) from human and yeast interacts with human p53. TBP co-immunoprecipitates with wild-type or mutant human p53 when incubated with the p53-specific monoclonal antibody and Protein A-agarose. Wild-type murine p53 has also been found to interact with human TBP. Protein blot assays have demonstrated that the interaction between p53 and human TBP is direct. By gel retention analysis, we have shown that the complex of TBP and p53 (both wild type and mutant) can bind to the TATA box. The similar qualitative binding capability of wild-type and mutant p53 with human TBP and the similarity of the two complexes in binding to the TATA box suggest that the functional discrimination between wild-type and mutant p53 may not lie in their ability to bind TBP. The nature of the p53.TBP or p53.TBP.TATA complex may determine the success of transcription. PMID: 8514746 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: Mol Cell Biol. 1993 Jun;13(6):3291-300. The p53 activation domain binds the TATA box-binding polypeptide in Holo-TFIID, and a neighboring p53 domain inhibits transcription. Liu X, Miller CW, Koeffler PH, Berk AJ. Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024-1570. Antioncogene product p53 is a transcriptional transactivator. To investigate how p53 stimulates transcription, we examined the interaction of p53 with general transcription factors in vitro. We found that p53 binds directly to the human TATA box-binding polypeptide (TBP). We also observed a direct interaction between p53 and purified holo-TFIID, a complex composed of TBP and a group of TBP-associated polypeptides known as TAFs. The p53 binding domain on TBP was mapped to the conserved region of TBP, including residues 220 to 271. The TBP binding domain on p53 was mapped to the p53 activation domain between residues 20 and 57. To analyze the significance of the p53-TBP interaction in p53 transactivation, we compared the ability of Gal4-p53 fusion proteins to bind to TBP in vitro and to activate transcription in transient transfection assays. Fusion proteins which bound to TBP activated transcription, and those that did not bind to TBP did not activate transcription to a detectable level, suggesting that a direct interaction between TBP and p53 is required for p53 transactivation. We also found that inclusion of residues 93 to 160 of p53 in a Gal4-p53 fusion repressed transcriptional activation 100-fold. Consequently, this region of p53 inhibits transcriptional activation by the minimal p53 activation domain. Highest levels of activation were observed with sequences 1 to 92 of p53 fused to Gal4, even though this construct bound to TBP in vitro with an affinity similar to that of other Gal4-p53 fusion proteins. We conclude that TBP binding is necessary for p53 transcriptional activation and that p53 sequences outside the TBP binding domain modulate the level of activation. PMID: 8497252 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: Oncogene. 1993 May;8(5):1183-93. Wild-type but not mutant p53 can repress transcription initiation in vitro by interfering with the binding of basal transcription factors to the TATA motif. Ragimov N, Krauskopf A, Navot N, Rotter V, Oren M, Aloni Y. Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, Israel. It has previously been shown that excess wild type (wt) p53 can repress the transcriptional activity of a variety of promoters in intact cells. To determine whether this transcriptional repression represented a direct effect of p53, wt and mutant p53 were prepared from E. coli-produced p53 and from insect cells infected with a recombinant baculovirus. When added into an in vitro transcription system, wt p53, but not mutant p53 reduced markedly transcription from the c-myc promoter, as well as from an array of other promoters, with the exception of an MHC class I gene promoter. The presence of wt p53 seemed to affect specifically the formation of the transcription preinitiation complex because preformed initiation complexes were completely refractory to wt p53, as was also the process of transcript elongation. Wild-type but not mutant p53 interfered with the stable binding of TBP and TFIIA to the TATA motif, although both wt and mutant p53 could associate in vitro with purified TBP. We propose that upon binding to TBP, wt but not mutant p53 specifically blocks the ability of TBP to engage in interactions required for efficient transcriptional initiation. This may account, at least in part, for the ability of excess wt p53 to inhibit cell proliferation and to interfere with neoplastic processes. PMID: 8479742 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: J Biol Chem. 1993 Feb 5;268(4):2284-7. Direct interaction between the transcriptional activation domain of human p53 and the TATA box-binding protein. Truant R, Xiao H, Ingles CJ, Greenblatt J. Banting and Best Institute of Medical Research, University of Toronto, Ontario, Canada. The human p53 tumor suppressor gene product can activate transcription by RNA polymerase II in the yeast, Saccharomyces cerevisiae, as well as in human cells. Several viral transcriptional activator proteins have been shown to directly contact TBP, the TATA box-binding subunit of the general initiation factor, TFIID. In this report, we use protein affinity chromatography to show that the cellular transcription factor, p53, interacts directly and specifically with yeast TBP. The TBP binding domain of p53 was localized to its N-terminal 73 amino acids. This highly acidic portion of p53 functions as a transcriptional activation domain and is deleted in some tumors induced by the Friend leukemia virus. A human tumor-derived oncogenic point mutation of p53, which lies outside the activation domain of p53, but reduces its ability to activate transcription, greatly reduced the ability of p53 to bind yeast TBP in vitro. This mutation probably affects the overall conformation of the protein and indirectly interferes with the ability of p53 to contact TBP and activate transcription. In contrast, a mutated oncogenic form of p53 that is unaffected in its ability to activate transcription bound yeast TBP as well as wild type p53. The human TBP activity in a HeLa extract also bound to the activation domain of p53. Our data support a general model in which DNA-bound activator proteins activate transcription by interacting with TBP. PMID: 8428901 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):12028-32. Wild-type p53 binds to the TATA-binding protein and represses transcription. Seto E, Usheva A, Zambetti GP, Momand J, Horikoshi N, Weinmann R, Levine AJ, Shenk T. Howard Hughes Medical Institute, Princeton University, NJ 08544-1014. p53 activates transcription of genes with a p53 response element, and it can repress genes lacking the element. Here we demonstrate that wild-type but not mutant p53 inhibits transcription in a HeLa nuclear extract from minimal promoters. Wild-type but not mutant p53 binds to human TATA-binding protein (TBP). p53 does not bind to yeast TBP, and it cannot inhibit transcription in a HeLa extract where yeast TBP substitutes for human TBP. These results suggest a model in which p53 binds to TBP and interferes with transcriptional initiation. PMID: 1465435 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------