1: Genes Dev. 2005 Apr 15;19(8):933-41. Epub 2005 Apr 1. The nucleolus as a stress sensor: JNK2 inactivates the transcription factor TIF-IA and down-regulates rRNA synthesis. Mayer C, Bierhoff H, Grummt I. Division of Molecular Biology of the Cell II, German Cancer Research Center, D-69120 Heidelberg, Germany. Christine.Mayer@dkfz.de Cells respond to a variety of extracellular and intracellular forms of stress by down-regulating rRNA synthesis. We have investigated the mechanism underlying stress-dependent inhibition of RNA polymerase I (Pol I) transcription and show that the Pol I-specific transcription factor TIF-IA is inactivated upon stress. Inactivation is due to phosphorylation of TIF-IA by c-Jun N-terminal kinase (JNK) at a single threonine residue (Thr 200). Phosphorylation at Thr 200 impairs the interaction of TIF-IA with Pol I and the TBP-containing factor TIF-IB/SL1, thereby abrogating initiation complex formation. Moreover, TIF-IA is translocated from the nucleolus into the nucleoplasm. Substitution of Thr 200 by valine as well as knock-out of Jnk2 prevent inactivation and translocation of TIF-IA, leading to stress-resistance of Pol I transcription. Our data identify TIF-IA as a downstream target of the JNK pathway and suggest a critical role of JNK2 to protect rRNA synthesis against the harmful consequences of cellular stress. PMID: 15805466 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Virology. 2003 Sep 15;314(1):423-31. The adeno-associated virus major regulatory protein Rep78-c-Jun-DNA motif complex modulates AP-1 activity. Prasad CK, Meyers C, Zhan DJ, You H, Chiriva-Internati M, Mehta JL, Liu Y, Hermonat PL. Department of Internal Medicine, Gene Therapy Center for Molecular Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV) infection. Mechanisms for this correlation may be by Rep78's (AAV's major regulatory protein) ability to bind the HPV-16 p97 promoter DNA and inhibit transcription, to bind and interfere with the functions of the E7 oncoprotein of HPV-16, and to bind a variety of HPV-important cellular transcription factors such as Sp1 and TBP. c-Jun is another important cellular factor intimately linked to the HPV life cycle, as well as keratinocyte differentiation and skin development. Skin is the natural host tissue for both HPV and AAV. In this article it is demonstrated that Rep78 directly interacts with c-Jun, both in vitro and in vivo, as analyzed by Western blot, yeast two-hybrid cDNA, and electrophoretic mobility shift-supershift assay (EMSA supershift). Addition of anti-Rep78 antibodies inhibited the EMSA supershift. Investigating the biological implications of this interaction, Rep78 inhibited the c-Jun-dependent c-jun promoter in transient and stable chloramphenicol acetyl-transferase (CAT) assays. Rep78 also inhibited c-Jun-augmented c-jun promoter as well as the HPV-16 p97 promoter activity (also c-Jun regulated) in in vitro transcription assays in T47D nuclear extracts. Finally, the Rep78-c-Jun interaction mapped to the amino-half of Rep78. The ability of Rep78 to interact with c-Jun and down-regulate AP-1-dependent transcription suggests one more mechanism by which AAV may modulate the HPV life cycle and the carcinogenesis process. PMID: 14517094 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Mol Cell Biol. 2003 Apr;23(8):2749-61. NF-kappa B-dependent assembly of an enhanceosome-like complex on the promoter region of apoptosis inhibitor Bfl-1/A1. Edelstein LC, Lagos L, Simmons M, Tirumalai H, Gelinas C. Center for Advanced Biotechnology and Medicine and Graduate Program in Biotechnology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA. Expression of the prosurvival Bcl-2 homologue Bfl-1/A1 is induced by NF-kappa B-activating stimuli, while B and T cells from c-rel knockout mice show an absolute defect in bfl-1/a1 gene activation. Here, we demonstrate NF-kappa B-dependent assembly of an enhanceosome-like complex on the promoter region of bfl-1. Binding of NF-kappa B subunit c-Rel to DNA nucleated the concerted binding of transcription factors AP-1 and C/EBP beta to the 5'-regulatory region of bfl-1. Optimal stability of the complex was dependent on proper orientation and phasing of the NF-kappa B site. Chromatin immunoprecipitation analyses demonstrated that T-cell activation triggers in vivo binding of endogenous c-Rel, c-Jun, C/EBP beta, and HMG-IC to the bfl-1 regulatory region, coincident with selective recruitment of coactivators TAFII250 and p300, SWI/SNF chromatin remodeling factor component BRG-1, and basal transcription factors TATA-binding protein (TBP) and TFIIB, as well as hyperacetylation of histones H3 and H4. These results highlight a critical role for NF-kappa B in bfl-1 transcription and point to the need for a complex and precise regulatory network to control bfl-1 expression. To our knowledge, this is the first demonstration of enhanceosome-mediated regulation of a cell death inhibitor. PMID: 12665576 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: J Biol Chem. 2001 Sep 7;276(36):33826-32. Epub 2001 Jul 13. The absence of activator protein 1-dependent gene expression in THP-1 macrophages stimulated with phorbol esters is due to lack of p38 mitogen-activated protein kinase activation. Carter AB, Tephly LA, Hunninghake GW. University of Iowa College of Medicine and the Iowa City Veterans Affairs Medical Center, Iowa City, Iowa 52242, USA. aaron-carter@uiowa.edu Activator protein 1 (AP-1) binds to the promoters of many genes involved in immune and inflammatory responses. We have previously shown that the p38 mitogen-activated protein (MAP) kinase regulates NF-kappa B-dependent gene expression by modulating the phosphorylation and subsequent activation of TATA-binding protein (TBP). In this study, we asked whether the p38 MAP kinase regulated the transcriptional activity of AP-1. We found that phorbol 12-myristate 13-acetate (PMA) was unable to drive the AP-1-dependent reporter gene in THP-1 cells. PMA activated both the extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase MAP kinases, but it did not activate the p38 MAP kinase. We found that cells expressing MAP kinase kinase 6(Glu), which is the upstream kinase that activates the p38 MAP kinase, had significantly increased AP-1-dependent gene expression alone and when stimulated with PMA. These cells also had increased phosphorylation of native c-Jun, suggesting that both c-Jun NH(2)-terminal kinase and p38 MAP kinases phosphorylate c-Jun. More importantly, expression of a constitutive active MAP kinase kinase 6(Glu) resulted in the phosphorylation of a His-TBP fusion protein and increased direct interaction of TBP with c-Jun. These findings suggest that in macrophages, the p38 MAP kinase regulates AP-1-driven transcription by modulating the activation of TBP. PMID: 11454854 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Cytokine Growth Factor Rev. 2001 Jun-Sep;12(2-3):189-205. X protein of hepatitis B virus modulates cytokine and growth factor related signal transduction pathways during the course of viral infections and hepatocarcinogenesis. Diao J, Garces R, Richardson CD. Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9. Hepatitis B virus produces chronic infections of the liver leading to cirrhosis and hepatocellular carcinoma. The X protein of hepatitis B virus (HBx) is a multifunctional protein that can interact with p53 but can also influence a variety of signal transduction pathways within the cell. In most instances this small viral protein favors cell survival and probably initiates hepatocarcinogenesis. HBx upregulates the activity of a number of transcription factors including NF-kappa B, AP-1, CREB, and TBP. However, the majority of HBx is localized to the cytoplasm where it interacts with and stimulates protein kinases such as protein kinase C, Janus kinase/STAT, IKK, PI-3-K, stress-activated protein kinase/Jun N-terminal kinase, and protein kinase B/Akt. This small viral protein can localize to the mitochondrion. HBx may act as an adaptor or kinase activator to influence signal transduction pathways. This review will attempt to analyze the involvement of HBx in signal transduction pathways during hepatitis B viral infections and hepatocellular carcinoma development. Publication Types: Review PMID: 11325602 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: J Biol Chem. 2001 Jul 6;276(27):25582-8. Epub 2001 Apr 20. c-Jun binds the N terminus of human TAF(II)250 to derepress RNA polymerase II transcription in vitro. Lively TN, Ferguson HA, Galasinski SK, Seto AG, Goodrich JA. Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA. c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250. The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro. PMID: 11316804 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: J Hum Virol. 2000 May-Jun;3(3):113-24. Binding of the human papillomavirus type 16 E7 oncoprotein and the adeno-associated virus Rep78 major regulatory protein in vitro and in yeast and the potential for downstream effects. Hermonat PL, Santin AD, Zhan D. Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock 72205, USA. OBJECTIVE: Both human papillomavirus (HPV) and adeno-associated virus (AAV) are common anogenital viruses and likely co-infect the epithelium in vivo. However, whereas HPVs are positively associated with cervical cancer, AAV appears to be negatively associated. In tissue culture, AAV-encoded Rep78--which is essential for AAV--inhibits gene expression and oncogenic transformation by HPV-16/18 and bovine papillomavirus type 1. Here we observed whether the HPV-16 E7 oncoprotein might recognize and bind Rep78. Further, upon finding Rep78-E7 binding, we investigated some of the potential downstream effects such an interaction might have. E7 is capable of recognizing a variety of proteins, including RB105, TATA box-binding protein (TBP), TBP-associated factor (TAF)(II)110, E2F, cyclins A and D, and c-jun. Some of these interactions are likely responsible for E7's cancer-promoting activity. STUDY DESIGN/METHODS: Rep78-E7 interaction was investigated in vitro by West(far)-Western and affinity chromatography analysis and in vivo in living yeast by the GAL4 two-hybrid cDNA assay. Mapping of the E7 binding domain within Rep78 was carried out using a series of amino- and carboxy-truncated Rep78 proteins in a West(far)-Western assay. Downstream effects of the interaction were analyzed by competitive affinity chromatography (protein-protein) and competitive electrophoretic mobility shift assay (protein-DNA). RESULTS: E7 and Rep78 were found to interact both in vitro and in vivo (yeast) in all assays attempted. The E7 binding domain within Rep78 was found to reside within amino acids 121-370. Regarding downstream effects of this interaction, Rep78 was found to mildly inhibit E7-TAF(II)110 and E7-RB105 interaction in vitro but to have little affect on E7-TBP interaction. Finally, it was found that E7 was able to affect Rep78's interaction with AAV's terminal repeat (TR) DNA in vitro, reducing the formation of the largest sized Rep78-TR complexes in a dosage-dependent manner. CONCLUSIONS: These data suggest that the Rep78-E7 interaction may have repercussions for both viruses. The Rep78-E7 interaction may be a second mechanism, in addition to Rep78 regulation of the p97 promoter, by which AAV inhibits HPV-16 oncogenic transformation. These data also suggest that HPV-16 may affect the AAV life cycle by altering Rep78-TR interaction. PMID: 10881991 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: J Biol Chem. 1999 Oct 22;274(43):30858-63. The p38 mitogen-activated protein kinase is required for NF-kappaB-dependent gene expression. The role of TATA-binding protein (TBP). Carter AB, Knudtson KL, Monick MM, Hunninghake GW. University of Iowa College of Medicine, Iowa City Veterans Administration Medical Center, Iowa City, Iowa 52242, USA. Endotoxin-induced cytokine gene transcription in monocytes and macrophages is regulated in part by NF-kappaB. We have previously shown that the p38 mitogen-activated protein (MAP) kinase is necessary for endotoxin-induced cytokine gene transcription. Due to the fact that most cytokine promoter sequences have active NF-kappaB sites, we hypothesized that the p38 MAP kinase was necessary for NF-kappaB-dependent gene expression. We found that NF-kappaB-dependent gene expression was reduced to near control levels with either SB 203580 or a dominant-negative p38 MAP kinase expression vector. Inhibition of the p38 MAP kinase did not alter NF-kappaB activation at any level, but it significantly reduced the DNA binding of TATA-binding protein (TBP) to the TATA box. The dominant-negative p38 MAP kinase expression vector interfered with the direct interaction of native TFIID (TBP) with a co-transfected p65 fusion protein. Likewise, this dominant-negative plasmid also interfered with the direct interaction of a co-transfected TBP fusion protein with the native p65 subunit. The p38 kinase also phosphorylated TFIID (TBP) in vitro, and SB 203580 inhibited phosphorylation of TFIID (TBP) in vivo. Thus, the p38 MAP kinase regulates NF-kappaB-dependent gene transcription, in part, by modulating activation of TFIID (TBP). PMID: 10521478 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: J Biol Chem. 1999 Apr 16;274(16):11115-24. SPI-B activates transcription via a unique proline, serine, and threonine domain and exhibits DNA binding affinity differences from PU.1. Rao S, Matsumura A, Yoon J, Simon MC. Department of Pathology, the University of Chicago, Chicago, Illinois 60637, USA. SPI-B is a B lymphocyte-specific Ets transcription factor that shares a high degree of similarity with PU.1/SPI-1. In direct contrast to PU.1(-/-) mice that die in utero and lack monocytes, neutrophils, B cells, and T cells, Spi-B-/- mice are viable and exhibit a severe B cell proliferation defect. Since PU.1 is expressed at wild type levels in Spi-B-/- B cells, the mutant mice provide genetic evidence that SPI-B and PU.1 have at least some non-redundant roles in B lymphocytes. To begin to understand the molecular basis for these defects, we delineated functional domains of SPI-B for comparison to those of PU.1. By using a heterologous co-transfection system, we identified two independent transactivation domains in the N terminus of SPI-B. Interestingly, only one of these domains (amino acids 31-61), a proline/serine/threonine-rich region, unique among Ets proteins, is necessary for transactivation of the immunoglobulin lambda light chain enhancer. This transactivation motif is in marked contrast to PU.1, which contains acidic and glutamine-rich domains. In addition, we describe a functional PU.1 site within the c-FES promoter which SPI-B fails to bind efficiently and transactivate. Finally, we show that SPI-B interacts with the PU.1 cofactors Pip, TBP, c-Jun and with lower affinity to nuclear factor interleukin-6beta and retinoblastoma. Taken together, these data suggest that SPI-B binds DNA with a different affinity for certain sites than PU.1 and harbors different transactivation domains. We conclude that SPI-B may activate unique target genes in B lymphocytes and interact with unique, although currently unidentified, cofactors. PMID: 10196196 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: J Virol. 1999 Jan;73(1):37-45. Targeting of the visna virus tat protein to AP-1 sites: interactions with the bZIP domains of fos and jun in vitro and in vivo. Morse BA, Carruth LM, Clements JE. Division of Comparative Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. The visna virus Tat protein is required for efficient viral transcription from the visna virus long terminal repeat (LTR). AP-1 sites within the visna virus LTR, which can be bound by the cellular transcription factors Fos and Jun, are also necessary for Tat-mediated transcriptional activation. A potential mechanism by which the visna virus Tat protein could target the viral promoter is by protein-protein interactions with Fos and/or Jun bound to AP-1 sites in the visna virus LTR. Once targeted to the visna virus promoter, the Tat protein could then interact with basal transcription factors to activate transcription. To examine protein-protein interactions with cellular proteins at the visna virus promoter, we used an in vitro protein affinity chromatography assay and electrophoretic mobility shift assay, in addition to an in vivo two-hybrid assay, to show that the visna virus Tat protein specifically interacts with the cellular transcription factors Fos and Jun and the basal transcription factor TBP (TATA binding protein). The Tat domain responsible for interactions with Fos and Jun was localized to an alpha-helical domain within amino acids 34 to 69 of the protein. The TBP binding domain was localized to amino acids 1 to 38 of Tat, a region previously described by our laboratory as the visna virus Tat activation domain. The bZIP domains of Fos and Jun were found to be important for the interactions with Tat. Mutations within the basic domains of Fos and Jun abrogated binding to Tat in the in vitro assays. The visna virus Tat protein was also able to interact with covalently cross-linked Fos and Jun dimers. Thus, the visna virus Tat protein appears to target AP-1 sites in the viral promoter in a mechanism similar to the interaction of human T-cell leukemia virus type 1 Tax with the cellular transcription factor CREB, by binding the basic domains of an intact bZIP dimer. The association between Tat, Fos, and Jun would position Tat proximal to the viral TATA box, where the visna virus Tat activation domain could contact TBP to activate viral transcription. PMID: 9847304 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: J Biol Chem. 1996 Nov 8;271(45):28138-45. CREB-binding protein activates transcription through multiple domains. Swope DL, Mueller CL, Chrivia JC. Department of Pharmacological and Physiological Sciences, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA. CREB-binding protein (CBP) functions as a coactivator molecule for a number of transcription factors including CREB, c-Fos, c-Jun, c-Myb, and several nuclear receptors. Although binding sites for these factors within CBP have been identified, the regions of CBP responsible for transcriptional activation are unknown. In this report, we show that the N-terminal half of CBP is sufficient for activation of CREB-mediated transcription and that this region contains a strong transcriptional activation domain (TAD). Both deletion of this TAD or sequestering of factors that the TAD binds using a squelching assay were found to greatly decrease the ability of CBP to activate CREB-mediated transcription. In vivo studies by others have shown that p300/CBP associates with TBP; using an in vitro approach, we show the N-terminal TAD binds TBP. We also examined the ability of the C terminus of CBP to activate transcription using GAL-CBP chimeras. With this approach, we identified two C-terminal TADs located adjacent to the c-Fos binding site. In previous studies, cAMP-dependent protein kinase A (PKA) increased the transcriptional activity of a GAL full-length CBP chimera in F9 cells, and of the C terminus in PC-12 cells. Here, we demonstrate that PKA also increased the ability of the N-terminal TADs of CBP to activate transcription in PC-12 but not F9 or COS-7 cells, suggesting that this PKA-responsiveness is cell type-specific. PMID: 8910428 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Genes Cells. 1996 Jul;1(7):687-705. D-type cyclin expression is decreased and p21 and p27 CDK inhibitor expression is increased when tsBN462 CCG1/TAFII250 mutant cells arrest in G1 at the restrictive temperature. Sekiguchi T, Noguchi E, Hayashida T, Nakashima T, Toyoshima H, Nishimoto T, Hunter T. Molecular Biology and Virology Laboratory, Salk Institute, La Jolla, CA 92037, USA. BACKGROUND: The tsBN462 temperature-sensitive mutant hamster cell line exhibits cell cycle arrest and apoptosis at the restrictive temperature of 39.5 degrees C, due to a point mutation in the CCG1/TAFII250 gene, which encodes a component of the general transcription factor TFIID. RESULTS: We now report that CCG1/TAFII250 persisted as a complex with TBP and associated proteins (TAFs) in tsBN462 cells at the restrictive temperature. FACScan analysis revealed that the tsBN462 mutation resulted in a failure to progress out of G0 into G1. Using two-dimensional gel electrophoresis we observed a decrease in the synthesis of several proteins, starting in the middle of the G1 phase, becoming very pronounced during late G1. The expression of the immediate early genes c-fos, c-jun and c-myc was normally induced by serum treatment of quiescent cells at the restrictive temperature, whereas expression of cyclins A, D1 and D3 was reduced. Expression of the cyclin-dependent kinase (CDK) inhibitor proteins p21 and p27 was enhanced. Consistent with the decreased cyclin D and increased p21/p27 expression, we found that phosphorylation of Rb was decreased at 39.5 degrees C. Cyclin A-, E- and Cdk2-associated histone H1 kinase activity was reduced concomitantly with the increase in p21 protein. CONCLUSION: Decreased cyclin/Cdk kinase activity and decreased Rb phosphorylation are possible causes of G1 cell cycle arrest in tsBN462 cells at the restrictive temperature. PMID: 9078394 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: EMBO J. 1996 Jan 15;15(2):319-33. GATA transcription factors associate with a novel class of nuclear bodies in erythroblasts and megakaryocytes. Elefanty AG, Antoniou M, Custodio N, Carmo-Fonseca M, Grosveld FG. National Institute for Medical Research, UK. The nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA-1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse nucleoplasmic localization. These foci, which were preferentially found adjacent to nucleoli or at the nuclear periphery, did not represent sites of active transcription or binding of GATA-1 to consensus sites in the beta-globin loci. Immunoelectron microscopy demonstrated the presence of intensely labelled structures likely to represent the GATA-1 foci seen by immunofluorescence. The GATA-1 nuclear bodies differed from previously described nuclear structures and there was no co-localization with nuclear antigens involved in RNA processing or other ubiquitous (Spl, c-Jun and TBP) or haemopoietic (NF-E2) transcription factors. Interestingly, GATA-2 and GATA-3 proteins also localized to the same nuclear bodies in cell lines co-expressing GATA-1 and -2 or GATA-1 and -3 gene products. This pattern of distribution is, thus far, unique to the GATA transcription factors and suggests a protein-protein interaction with other components of the nuclear bodies via the GATA zinc finger domain. PMID: 8617207 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Connect Tissue Res. 1996;35(1-4):23-31. Characterization of the bone sialoprotein (BSP) gene promoter. Sodek J, Li JJ, Kim RH, Ogata Y, Yamauchi M. MRC Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Ont., Canada. Bone sialoprotein is a 34 kDa phosphorylated and sulphated glycoprotein that is essentially unique to mineralizing connective tissues. Recent studies on the developmental expression of BSP mRNA and the temporo-spatial appearance of the protein during bone formation in vivo and in vitro have demonstrated that BSP is expressed by differentiated osteoblasts and that it may function in the initial nucleation of hydroxyapatite crystals in de novo bone formation. To study the cell-specific regulation of BSP we have isolated genomic clones that encompass the BSP promoter regions of both the human and rat genes. These promoters are characterized by a highly conserved region (BSP Box) that extends upstream from the transcription start site to nt -370. Within this region the immediate promoter is further characterized by a unique inverted TATA box and an inverted CCAAT box, both of which are required for basal transcriptional activity. The TATA box is overlapped by a vitamin D3 response element (VDRE) which appears to mediate vitamin D suppression of BSP gene transcription by competing with the TATA-binding protein (TBP) for occupancy of the site of the pre-initiation complex formation. Mutation of the inverted TATA box into a normal TATA sequence increases transcription slightly but does not affect the functionality of the VDRE indicating that the orientation of the TATA box is not critical for these functions. Further upstream an AP-1 site, overlapped by a steroid hormone response-like sequence, mediates down-regulation of BSP transcription induced by TPA that is abrogated by a complex interaction between Jun and the glucocorticoid receptor protein induced by dexamethasone. Thus, the characterization of approximately 3 kb of the BSP promoter and approximately 2 kb of the first intron has revealed several sites of transcriptional regulation that are important in regulating BSP expression and, consequently, bone formation. PMID: 9084640 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: J Biol Chem. 1995 May 5;270(18):10754-63. Repression of the c-Jun trans-activation function by the adenovirus type 12 E1A 52R protein correlates with the inhibition of phosphorylation of the c-Jun activation domain. Brockmann D, Bury C, Kroner G, Kirch HC, Esche H. Institute of Molecular Biology (Cancer Research), University of Essen Medical School, Federal Republic of Germany. The early region 1A 52R polypeptide, a protein expressed exclusively by the in vivo oncogenic adenovirus subtype 12, represses the trans-activating function of the cellular transcription factor complex AP-1 consisting of c-Jun-c-Jun homodimers. In this report we demonstrate that the repression in vivo correlates with a direct physical interaction of the adenovirus protein with c-Jun in vitro. Interestingly, the 52R protein binds to the bZIP domain of c-Jun essential for dimerization and DNA binding but not to the c-Jun activation domain. This interaction does not prevent the promoter binding of c-Jun/AP-1. Moreover, the physical association between c-Jun and the TATA box-binding protein TBP is not disturbed by the 52R polypeptide. In fact, we show evidence that down-regulation of c-Jun activity by the adenoviral protein is due to the inhibition of phosphorylation of the c-Jun trans-activation domain. In vivo phosphorylation of the c-Jun activation domain is necessary for the interaction of c-Jun with specific cofactors such as CBP and therefore a prerequisite for the activation of target genes. Due to these results we propose a model in which the 52R protein represses the trans-activating function of c-Jun by preventing its phosphorylation through a specific kinase necessary for the activation of the cellular transcription factor. PMID: 7738014 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Biochem J. 1995 Feb 1;305 ( Pt 3):967-74. In vitro association between the Jun protein family and the general transcription factors, TBP and TFIIB. Franklin CC, McCulloch AV, Kraft AS. Division of Haematology and Oncology, University of Alabama at Birmingham 35294. Transcriptional activator proteins interact with the general transcription factors TATA-binding protein (TBP), TFIIB and/or other TBP-associated factors (TAFs). Using affinity chromatography we demonstrate that members of the Jun family of transcriptional activators interact with both TBP and TFIIB in vitro. TBP binds to both the N-terminal activation domain and C-terminal bZIP regions of c-Jun, whereas TFIIB binds to only the c-Jun bZIP domain. This interaction requires the dimerization of the Jun protein. The ability of the N-terminal activation domains of c-Jun, JunB, JunD and v-Jun to interact with TBP in vitro correlates with their transcriptional activity in vivo. Domain mapping experiments indicate that c-Jun interacts with the conserved C-terminus of TBP. Studies using a set of TFIIB inframe deletion mutants demonstrate that C-terminal amino acids 178-201 and 238-316 play an important role in modulating the interaction between TFIIB and c-Jun. Although phosphorylation of the c-Jun N-terminal activation domain stimulates c-Jun transcriptional activity in vivo, it has no effect on the ability of c-Jun to interact with either TBP or TFIIB in vitro. These data suggest that the Jun family of activator proteins may activate transcription by interacting with the general transcription factors TBP and TFIIB. PMID: 7848298 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: C R Acad Sci III. 1993 Dec;316(12):1424-32. The bZIP motif of the Epstein-Barr virus (EBV) transcription factor EB1 mediates a direct interaction with TBP. Mikaelian I, Manet E, Sergeant A. ENS-CNRS UMR49, Ecole Normale Superieure de Lyon, France. The EBV transcription factor EB1, is a key determinant of the switch from the latent infection to the lytic cycle. EB1 belongs to the Jun, Fos, ATF, CREB, C/EBP and GCN4 family of proteins, carrying a sequence-specific DNA-binding domain called "basic-Zipper" (bZIP). The N-terminal region of EB1 is required for transcriptional activation, whereas the C-terminal region contains the DNA-binding domain. The mechanism by which site-specific transcription factors increase specific initiation at polymerase II dependent promoters is thought to occur via recruitment and stabilization of components that form the initiation complex, i.e., TFIID, TFIIA, TFIIB, TFIIE, TFIIG, TFIIH, TFIIJ and pol II. TFIID is not a single protein but consists of the TATA-binding protein TBP plus several distinct and tightly associated proteins called TAFs. More specifically, in vitro studies have revealed that the TAFs are not required for basal transcription, but are essential for mediating regulated transcription by different upstream activators. TFIID binding at the promoter sites is one of the limiting steps in the assembly of the initiation complex. Direct interactions with TBP or with one or several TAFs, mediated by the activation domain of site specific activators, could influence the binding rate of TFIID, and thus provide one of the mechanisms by which transcription is regulated. We show here that EB1 interacts directly with TBP in vitro, and that it is the bZIP domain, likely the region contacting the DNA rather than the activation domain, which is required for physical contact between EB1 and TBP. PMID: 8087622 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Gene Expr. 1993;3(1):37-48. The bZIP domains of Fos and Jun mediate a physical association with the TATA box-binding protein. Ransone LJ, Kerr LD, Schmitt MJ, Wamsley P, Verma IM. Molecular Biology and Virology Laboratory, Salk Institute, San Diego, CA 85800. Fos and Jun oncoproteins form a complex that regulates transcription from promoters containing AP-1 binding sites. These two proteins, like other transcriptional activators, are likely to stimulate transcription through direct and/or indirect interactions with members of the basal transcriptional machinery. The ability of c-Fos and c-Jun proteins to interact directly with the TATA box-binding protein (TBP), the general transcription factor required for initiating the assembly of transcription complexes, was investigated. Using co-immunoprecipitation and protein-protein association assays, we show that both c-Fos and c-Jun bind specifically and stably to TBP. Mutational analysis demonstrates that both the basic region and leucine zipper domains of c-Fos and c-Jun are necessary and sufficient for stable association with TBP. A 51-residue region from the conserved C-terminal region of TBP, previously shown to be the binding site for the viral activator protein E1A, interacts with c-Fos and c-Jun proteins. We propose that c-Fos and c-Jun proteins function as transcriptional activators, in part by recruiting TBP to form complexes to initiate RNA synthesis. PMID: 7685215 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------