1: BMC Biochem. 2005 May 5;6(1):7. Stimulation of Myc transactivation by the TATA binding protein in promoter-reporter assays. Barrett JF, Lee LA, Dang CV. Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. jbarret1@jhmi.edu BACKGROUND: The c-Myc oncogenic transcription factor heterodimerizes with Max, binds specific DNA sites and regulates transcription. The role of Myc in transcriptional activation involves its binding to TRRAP and histone acetylases; however, Myc's ability to activate transcription in transient transfection assays is remarkably weak (2 to 5 fold) when compared to other transcription factors. Since a deletion Myc mutant D106-143 and a substitution mutant W135E that weakly binds TRRAP are still fully active in transient transfection reporter assays and the TATA binding protein (TBP) has been reported to directly bind Myc, we sought to determine the effect of TBP on Myc transactivation. RESULTS: We report here a potent stimulation of Myc transactivation by TBP, allowing up to 35-fold transactivation of reporter constructs. Although promoters with an initiator (InR) element briskly responded to Myc transactivation, the presence of an InR significantly diminished the response to increasing amounts of TBP. We surmise from these findings that promoters containing both TATA and InR elements may control Myc responsive genes that require brisk increased expression within a narrow window of Myc levels, independent of TBP. In contrast, promoters driven by the TATA element only, may also respond to modulation of TBP activity or levels. CONCLUSION: Our observations not only demonstrate that TBP is limiting for Myc transactivation in transient transfection experiments, but they also suggest that the inclusion of TBP in Myc transactivation assays may further improve the characterization of c-Myc target genes. PMID: 15876353 [PubMed - in process] --------------------------------------------------------------- 2: Mech Dev. 2005 Apr;122(4):545-56. Epub 2005 Jan 22. Pontin and Reptin regulate cell proliferation in early Xenopus embryos in collaboration with c-Myc and Miz-1. Etard C, Gradl D, Kunz M, Eilers M, Wedlich D. Zoologisches Institut II, Universitat Karlsruhe (TH), Kaiserstrasse 12, 76131 Karlsruhe, Germany. Pontin (Tip49) and Reptin (Tip48) are highly conserved components of multimeric protein complexes important for chromatin remodelling and transcription. They interact with many different proteins including TATA box binding protein (TBP), beta-catenin and c-Myc and thus, potentially modulate different pathways. As antagonistic regulators of Wnt-signalling, they control wing development in Drosophila and heart growth in zebrafish. Here we show that the Xenopus xPontin and xReptin in conjunction with c-Myc regulate cell proliferation in early development. Overexpression of xPontin or xReptin results in increased mitoses and bending of embryos, which is mimicked by c-Myc overexpression. Furthermore, the knockdown of either xPontin or xReptin resulted in embryonic lethality at late gastrula stage, which is abrogated by the injection of c-Myc-RNA. The N-termini of xPontin and xReptin, which mediate the mitogenic effect were mapped to contain c-Myc interaction domains. c-Myc protein promotes cell cycle progression either by transcriptional activation through the c-Myc/Max complex or by repression of cyclin dependent kinase inhibitors (p21, p15) through c-Myc/Miz-1 interaction. Importantly, xPontin and xReptin exert their mitogenic effect through the c-Myc/Miz-1 pathway as dominant negative Miz-1 and wild-type c-Myc but not a c-Myc mutant deficient in Miz-1 binding could rescue embryonic lethality. Finally, promoter reporter studies revealed that xPontin and xReptin but not the N-terminal deletion mutants enhance p21 repression by c-Myc. We conclude that xPontin and xReptin are essential genes regulating cell proliferation in early Xenopus embryogenesis through interaction with c-Myc. We propose a novel function of xPontin and xReptin as co-repressors in the c-Myc/Miz-1 pathway. PMID: 15804567 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: J Mol Biol. 2005 Feb 11;346(1):175-89. Epub 2004 Dec 10. N and C-terminal sub-regions in the c-Myc transactivation region and their joint role in creating versatility in folding and binding. Fladvad M, Zhou K, Moshref A, Pursglove S, Safsten P, Sunnerhagen M. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden. The proto-oncogene c-myc governs the expression of a number of genes targeting cell growth and apoptosis, and its expression levels are distorted in many cancer forms. The current investigation presents an analysis by proteolysis, circular dichroism, fluorescence and Biacore of the folding and ligand-binding properties of the N-terminal transactivation domain (TAD) in the c-Myc protein. A c-Myc sub-region comprising residues 1-167 (Myc1-167) has been investigated that includes the unstructured c-Myc transactivation domain (TAD, residues 1-143) together with a C-terminal segment, which appears to promote increased folding. Myc1-167 is partly helical, binds both to the target proteins Myc modulator-1 (MM-1) and TATA box-binding protein (TBP), and displays the characteristics of a molten globule. Limited proteolysis divides Myc1-167 in two halves, by cleaving in a predicted linker region between two hotspot mutation regions: Myc box I (MBI) and Myc box II (MBII). The N-terminal half (Myc1-88) is unfolded and does not alone bind to target proteins, whereas the C-terminal half (Myc92-167) has a partly helical fold and specifically binds both MM-1 and TBP. Although this might suggest a bipartite organization in the c-Myc TAD, none of the N and C-terminal fragments bind target protein with as high affinity as the entire Myc1-167, or display molten globule properties. Furthermore, merely linking the MBI with the C-terminal region, in Myc38-167, is not sufficient to achieve binding and folding properties as in Myc1-167. Thus, the entire N and C-terminal regions of c-Myc TAD act in concert to achieve high specificity and affinity to two structurally and functionally orthogonal target proteins, TBP and MM-1, possibly through a mechanism involving molten globule formation. This hints towards understanding how binding of a range of targets can be accomplished to a single transactivation domain. PMID: 15663936 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Int J Mol Med. 2004 Oct;14(4):497-504. The influence of neural signal transduction on EEC gene expression under consideration of chromatin, following myenteric ablation (review). Holle GE. Gastroenterology Research Laboratory, University Policlinic, Walther Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians-University, Pettenkoferstrasse 8a, Munich D-80336, Germany. hollegresearch@aol.com Significant numerical and spatial changes in 5-HT i.r. cells, CCK i.r. I-cells, glucagon and glicentin i.r. I-cells, somatostatin i.r. D-cells and neurotensin i.r. N-cells occur after a 98% myenteric ablation in the rat. Signal transduction from G-protein-coupled crypt cell receptors (m2, m3; VCAP1 and 2, CAP1; Y2, Y5, Y4) stimulates a cAMP-responsive transcription machinery in which phosphorylation of the cAMP-responsive elements (e.g. CREB) is the first step in initiation of transcription. A DNA pre-initiation complex (PIC), consisting of DNA transcription activators, general activators (TFIID, IIA, IIB, IIF, IIE, II-I and IIH), at least 14 different TAFIIs and CBP/300 coactivators which contain multiple enzymatic activities, associated with the central TBP (TATA-binding protein), which together bind to the RNA-polymerase II holoenzyme disrupts chromatin blockade over the promoter with or without the intervention of activated chromatin remodeling factors. CBP/p300 contains several highly conserved domains e.g., KIX, whose methylation by CARM-1 represses CREB transcription activation, but the bromo-binding domain of CBP increases CREB transcription. A similar positive/negative switch occurs in the regulation of gastrointestinal hormones by transcription factors, from Myc/Max to Mad/Max + corepressor mSin3A, during terminal differentiation of the cell. From these observations we conclude that the primary targets for neural signals are factors of the basal DNA transcriptional apparatus, whose promoter factors then activate chromatin induction, which facilitates transcription positively or negatively. Publication Types: Review Review, Tutorial PMID: 15375574 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Anal Biochem. 2004 May 15;328(2):101-8. Gene expression analysis by real-time reverse transcription polymerase chain reaction: influence of tissue handling. Almeida A, Paul Thiery J, Magdelenat H, Radvanyi F. Laboratoire de Transfert, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France. Factors such as warm ischemia and time at room temperature before tissue treatment may influence the results of mRNA expression analyses on tissue specimens obtained during surgery. We evaluated the effect of these factors on RNA integrity and mRNA expression levels by incubating freshly obtained mouse liver tissue at 25 or 37 degrees C for periods of 0-4 h. Changes in the mRNA expression levels of seven genes, Tbp, Eef1a, Fos, Junb, Myc, Vegf, and Glut2, were determined by real-time reverse transcription-polymerase chain reaction. Incubation at 25 degrees C for up to 4 h only slightly altered (by a factor of less than 2) levels of mRNA for Tbp, Eef1a, Junb, Myc, Vegf, and Glut2. This result is consistent with limited RNA degradation at this temperature. Incubation at 37 degrees C strongly affected the levels of these mRNAs. Four hours of incubation at this temperature resulted in extensive RNA degradation, with mRNA levels falling to 1/10th those before incubation. When relative quantification was performed, i.e., quantification of the target gene transcripts in comparison to an endogenous housekeeping transcript (Tbp or Eef1a), the changes in mRNA levels were reduced to less than 2.5-fold. Fos behaved very differently from the other genes tested on incubation, with Fos mRNA levels increasing considerably following incubation at either 25 or 37 degrees C. Our data suggest that, with the exception of certain genes induced by tissue injury, relative quantification of mRNA, even on degraded RNA samples, can provide a reliable estimate of in vivo mRNA levels. PMID: 15113684 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Clin Cancer Res. 2003 Sep 1;9(10 Pt 1):3641-4. Enolase-alpha is frequently down-regulated in non-small cell lung cancer and predicts aggressive biological behavior. Chang YS, Wu W, Walsh G, Hong WK, Mao L. Molecular Biology Laboratory, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030, USA. PURPOSE: Enolase-alpha is a cytoplasmic glycolytic enzyme important in the formation of phosphoenolpyruvate. Enolase-alpha and c-myc binding protein (MBP-1) originate from a single gene through alternative use of translational starting sites. Both enolase-alpha and MBP-1 can bind to the P2 element in the c-myc promoter and compete with TATA-box binding protein (TBP) to suppress transcription of c-myc. EXPERIMENTAL DESIGN: To determine a potential role of enolase-alpha in vivo, we analyzed enolase-alpha expression in non-small cell lung cancer (NSCLC) tissues from 46 patients by Western blotting and immunohistochemical analysis. RESULTS: Twelve (26%) of the 46 tumors showed a significantly reduced enolase-alpha expression. Although no statistically significant association was observed between the down-regulation of enolase-alpha and pathological stage, tumor histology, or differentiation, the patients whose tumors showed reduced enolase-alpha expression had a significantly poorer overall survival compared with those without down-regulation of this molecule (P = 0.0398). CONCLUSIONS: Our results indicate down-regulation of enolase-alpha is common in NSCLC and may play an important role in lung tumorigenesis. PMID: 14506152 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: 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] --------------------------------------------------------------- 8: Dev Cell. 2002 Jun;2(6):807-17. Erratum in: Dev Cell 2002 Aug;3(2):299. An evolutionarily conserved mediator of plant disease resistance gene function is required for normal Arabidopsis development. Holt BF 3rd, Boyes DC, Ellerstrom M, Siefers N, Wiig A, Kauffman S, Grant MR, Dangl JL. Department of Biology, University of North Carolina, Chapel Hill, NC 27599, USA. Plants recognize many pathogens through the action of a diverse family of proteins called disease resistance (R) genes. The Arabidopsis R gene RPM1 encodes resistance to specific Pseudomonas syringae strains. We describe an RPM1-interacting protein that is an ortholog of TIP49a, previously shown to interact with the TATA binding protein (TBP) complex and to modulate c-myc- and beta-catenin-mediated signaling in animals. Reduction of Arabidopsis TIP49a (AtTIP49a) mRNA levels results in measurable increases of two R-dependent responses without constitutively activating defense responses, suggesting that AtTIP49a can act as a negative regulator of at least some R functions. Further, AtTIP49a is essential for both sporophyte and female gametophyte viability. Thus, regulators of R function overlap with essential modulators of plant development. PMID: 12062092 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Mol Cell. 2002 Mar;9(3):553-62. Nuclear receptor function requires a TFTC-type histone acetyl transferase complex. Yanagisawa J, Kitagawa H, Yanagida M, Wada O, Ogawa S, Nakagomi M, Oishi H, Yamamoto Y, Nagasawa H, McMahon SB, Cole MD, Tora L, Takahashi N, Kato S. Institute of Molecular and Cellular Biosciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Nuclear receptors (NRs) regulate transcription in a ligand-dependent way through two types of coactivator complexes: the p160/CBP histone acetyl transferase (HAT) complex and the DRIP/TRAP/SMCC complex without HAT activity. Here we identified a large human (h) coactivator complex necessary for the estrogen receptor alpha (ERalpha) transactivation. This complex contains the GCN5 HAT, the c-Myc interacting protein TRRAP/PAF400, TAF(II)30, and other subunits. Similarly to known TFTC (TBP-free TAF(II)-containing)-type HAT complexes (hTFTC, hPCAF, and hSTAGA), TRRP directly interacted with liganded ER alpha, or other NRs. ER alpha transactivation was enhanced by the purified complex in vitro. Antisense TRRAP RNA inhibited estrogen-dependent cell growth of breast cancer cells. Thus, the isolated TFTC-type HAT complex acts as a third class of coactivator complex for NR function. PMID: 11931763 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Genome. 2001 Dec;44(6):1100-3. TAFII70 protein in Cajal bodies of the amphibian germinal vesicle. Bucci S, Giani L, Mancino G, Pellegrino M, Ragghianti M. Laboratorio di Biologia cellulare e dello sviluppo, Dipartimento di Fisiologia e Biochimica, Pisa, Italy. buccis@dfb.unipi.it The localization of the TATA-binding protein (TBP) associated factor II70 (TAFII70) in the germinal vesicle (GV) of newt oocytes was investigated. In spreads of GV content, anti-hTAFII70 monoclonal antibody (mAb) stained Cajal bodies (CBs) that were either attached to specific sites on the lampbrush chromosomes or free in the nucleoplasm. To confirm this localization the PwTAFII70 cDNA was cloned and myc-tagged transcripts injected into the oocyte cytoplasm. Newly translated PwTAFII70 protein was detected a few hours later in the Cajal bodies. These data support the hypothesis that Cajal bodies are the assembly sites of the transcription machinery of the oocyte nucleus. TAFII70 protein can play a role in lampbrush transcription; alternatively TAFII70 can be considered a component in the subset of TFIID complexes that do not function during oogenesis, but are accumulated in the oocyte for later use during early development. PMID: 11768213 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Oncogene. 2001 Sep 20;20(42):6039-47. In vivo complex formation of PU.1 with HDAC1 associated with PU.1-mediated transcriptional repression. Kihara-Negishi F, Yamamoto H, Suzuki M, Yamada T, Sakurai T, Tamura T, Oikawa T. Department of Cell Genetics, Sasaki Institute, 2-2, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. We previously reported that overexpression of PU.1, a member of the Ets family of transcription factors, induces differentiation inhibition and apoptosis associated with c-Myc down-regulation in murine erythroleukemia (MEL) cells. To understand the molecular mechanism by which c-Myc is down-regulated due to overexpression of PU.1, we performed luciferase reporter assays using the mouse c-myc promoter. PU.1 repressed the activities of not only the c-myc promoter but also several other promoters. Experiments with deletion mutants of PU.1 revealed that the C-terminal region spanning amino acids (aa) 123-272 including the PEST and ETS domains but not the activation domain was sufficient for this transcriptional repression. It was unlikely that the repression was due to sequestration of a limited amount of CBP/p300 nor pCAF, because overexpression of these co-activators did not relieve PU.1-mediated transcriptional repression. Instead, it was found that the C-terminal aa 101-272 of PU.1 formed a complex with mSin3A and HDAC1 in vivo, which was speculated to be associated with the repression. The C-terminal region of PU.1 also formed a complex with the basic transcription factor TBP in vitro and in vivo. Our results suggest that overexpression of PU.1 induces transcriptional repression in several gene promoters including the c-myc promoter which may be mediated by its complex formation with HDACs. PMID: 11593411 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: J Biol Chem. 2001 Oct 26;276(43):40127-32. Epub 2001 Aug 20. How transcriptional activators bind target proteins. Hermann S, Berndt KD, Wright AP. Department of Natural Sciences, Sodertorns hogskola, Box 4101, S-14104 Huddinge, Sweden. Stefan.Hermann@sh.se The product of the proto-oncogene c-myc influences many cellular processes through the regulation of specific target genes. Through its transactivation domain (TAD), c-Myc protein interacts with several transcription factors, including TATA-binding protein (TBP). We present data that suggest that in contrast to some other transcriptional activators, an extended length of the c-Myc TAD is required for its binding to TBP. Our data also show that this interaction is a multistep process, in which a rapidly forming low affinity complex slowly converts to a more stable form. The initial complex formation results from ionic or polar interactions, whereas the slow conversion to a more stable form is hydrophobic in nature. Based on our results, we suggest two alternative models for activation domain/target protein interactions, which together provide a single universal paradigm for understanding activator-target factor interactions. PMID: 11514548 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Microbiol Res. 2001;156(1):1-7. A novel surfactant nanoemulsion with a unique non-irritant topical antimicrobial activity against bacteria, enveloped viruses and fungi. Hamouda T, Myc A, Donovan B, Shih AY, Reuter JD, Baker JR Jr. Department of Internal Medicine, and Center for Biologic Nanotechnology, University of Michigan Medical School, Ann Arbor, USA. A novel non-ionic surfactant nanoemulsion designated 8N8 has been tested for its biocidal activity. One percent 8N8 produced effective bactericidal activity against Bacillus cereus, Bacillus subtilis, Haemophilus influenzae, Neisseria gonorrhoeae, Streptococcus pneumoniae, and Vibrio cholerae in 15 minutes. In contrast, most enteric gram-negative bacteria were resistant to 8N8. One percent 8N8 was also virucidal within 15 minutes for all tested enveloped viruses, including Herpes simplex type 1, influenza A and vaccinia viruses. One percent 8N8 also demonstrated fungistatic activity on Candida albicans. The rapid and non-specific inactivation of vegetative bacteria and enveloped viruses, in addition to its fungistatic activity and low toxicity in experimental animals, makes 8N8 a potential candidate for use as a topical biocidal agent. PMID: 11372645 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Oncogene. 2000 Nov 30;19(51):5944-50. Degradation of the E7 human papillomavirus oncoprotein by the ubiquitin-proteasome system: targeting via ubiquitination of the N-terminal residue. Reinstein E, Scheffner M, Oren M, Ciechanover A, Schwartz A. Department of Biochemistry and the Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Haifa, Israel. The E7 oncoprotein of the high risk human papillomavirus type 16 (HPV-16), which is etiologically associated with uterine cervical cancer, is a potent immortalizing and transforming agent. It probably exerts its oncogenic functions by interacting and altering the normal activity of cell cycle control proteins such as p21WAF1, p27KIP1 and pRb, transcriptional activators such as TBP and AP-1, and metabolic regulators such as M2-pyruvate kinase (M2-PK). Here we show that E7 is a short-lived protein and its degradation both in vitro and in vivo is mediated by the ubiquitin-proteasome pathway. Interestingly, ubiquitin does not attach to any of the two internal Lysine residues of E7. Substitution of these residues with Arg does not affect the ability of the protein to be conjugated and degraded; in contrast, addition of a Myc tag to the N-terminal but not to the C-terminal residue, stabilizes the protein. Also, deletion of the first 11 amino acid residues stabilizes the protein in cells. Taken together, these findings strongly suggest that, like MyoD and the Epstein Barr Virus (EBV) transforming Latent Membrane Protein 1 (LMPI), the first ubiquitin moiety is attached linearly to the free N-terminal residue of E7. Additional ubiquitin moieties are then attached to an internal Lys residue of the previously conjugated molecule. The involvement of E7 in many diverse and apparently unrelated processes requires tight regulation of its function and cellular level, which is controlled in this case by ubiquitin-mediated proteolysis. PMID: 11127826 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Int Immunol. 2000 Jul;12(7):1085-93. The TATA binding protein, c-Myc and survivin genes are not somatically hypermutated, while Ig and BCL6 genes are hypermutated in human memory B cells. Shen HM, Michael N, Kim N, Storb U. Departments of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. Immunoglobulin (IG:) genes are hypermutated in mature B cells after interaction with antigen and T cells in a germinal center reaction. We and others have recently shown that the human BCL6 gene is also hypermutated in human peripheral blood memory B cells and tonsils. A preliminary analysis of other non-Ig genes (c-MYC:, S14 and AFP) suggested that they were not mutated in memory B cells. We have now performed an in-depth analysis of three non-Ig genes that are expressed in germinal center B cells in two human donors in whom BCL6 is highly mutated. It was found that the TATA binding protein (TBP), c-MYC: and survivin genes are not hypermutated. This lack of targeting by the Ig hypermutation mechanism must be due to the lack of regulatory DNA elements, since the primary sequences of the three tested genes have at least as high intrinsic mutability indices as the BCL6 gene. PMID: 10882420 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Oncogene. 1999 Nov 18;18(48):6810-7. Nmi protein interacts with regions that differ between MycN and Myc and is localized in the cytoplasm of neuroblastoma cells in contrast to nuclear MycN. Bannasch D, Weis I, Schwab M. Division of Cytogenetics-H0400, Deutsches Krebsforschungszentrum, Heidelberg, Germany. Myc family proteins play an important role in cellular processes such as proliferation, differentiation, apoptosis and transformation. A number of interaction partners of Myc have been identified, such as Max, p107, TBP, YY1, Miz-1, AP-2 and Nmi. Both Max and Nmi also bind to MycN. In contrast to the well defined binding of Max to Myc family proteins the interaction of Nmi with Myc or MycN is only poorly characterized. By employing the yeast two-hybrid system we have mapped the regions of MycN and Myc responsible for binding to Nmi. For MycN exclusively a central region mediates binding to Nmi. In contrast, for Myc a C-terminal portion of the protein, and possibly also a central part, is involved in Nmi interaction. Nmi does not interact with Max and has no transactivation capabilities in yeast, suggesting that Nmi alone is not a transcriptional activator in mammalian cells. Immunofluorescence demonstrates that both in 293 embryonic kidney cells and in Kelly neuroblastoma cells all detectable ectopically expressed Nmi is localized in the cytoplasm, in part in a punctate, granular pattern. MycN, which is highly expressed in Kelly cells consequent to amplification, appears to be localized exclusively in the nuclei. This directly demonstrates that in the same cell at least the major proportion of MycN and Nmi is localized in different cellular compartments. This result is confirmed by the finding that endogenous Nmi, which is expressed in Kelly cells only after stimulation with interferon gamma, is detected exclusively in the cytoplasm of these cells. Therefore only a very small amount of MycN and Nmi is likely to be involved in MycN/Nmi interaction in vivo. PMID: 10597290 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Anticancer Drug Des. 1999 Jun;14(3):179-86. Effect of ecteinascidin-743 on the interaction between DNA binding proteins and DNA. Bonfanti M, La Valle E, Fernandez Sousa Faro JM, Faircloth G, Caretti G, Mantovani R, D'Incalci M. Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy. Ecteinascidin-743 (ET-743) is a tetrahydroisoquinoline alkaloid isolated from Ecteinascidia turbinata, a tunicate growing in mangrove roots in Caribbean. It has been shown to bind in the minor groove of DNA forming covalent adducts by reaction of the N2 of guanine with the carbinolamine moiety. We investigated ET-743 ability to inhibit the binding of different transcription factors to their consensus sequences by using gel shift assays. We have selected three types of factors: (i) oncogene products such as MYC, c-MYB and Maf; (ii) transcriptional activators regulated during the cell cycle as E2F and SRF; and (iii) general transcription factors such as TATA binding protein (TBP), Sp1 and NF-Y. We observed no inhibition of the binding of Sp1, Maf, MYB and MYC. Inhibition of DNA binding was observed for TBP, E2F, SRF at ET-743 concentrations ranging from 50 to 300 microM. The inhibition of binding of NF-Y occurs at even lower concentrations (i.e. 10-30 microM) when the recombinant subunits of NF-Y are preincubated with the drug, indicating that the inhibition of NF-Y binding does not require previous ET-743 DNA binding. Since NF-Y is a trimer containing two subunits with high resemblance to histones H2B and H2A, we have investigated the effect of ET-743 on nucleosome reconstitution. ET-743 caused a decrease of the nucleosomal band at 100 nM, with the complete disappearance of the band at 3-10 microM. These data suggest that the mode of action of this novel anticancer drug is related to its ability to modify the interaction between some DNA binding proteins and DNA. PMID: 10500494 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Oncogene. 1999 Aug 12;18(32):4598-605. Transcriptional regulation by targeted recruitment of cyclin-dependent CDK9 kinase in vivo. Majello B, Napolitano G, Giordano A, Lania L. Department of Genetics, Molecular and General Biology, University of Naples 'Federico II' and International Institute of Genetics and Biophysics, CNR, Naples, Italy. The CDK9 kinase in association with Cyclin T is a component of the transcription positive-acting complex pTEFb which facilitates the transition from abortive to productive transcription elongation by phosphorylating the carboxyl-terminal domain of RNA polymerase II. The Cyclin T1/CDK9 complex is implicated in Tat transactivation, and it has been suggested that Tat functions by recruiting this complex to RNAPII through cooperative binding to RNA. Here, we demonstrate that targeted recruitment of Cyclin T1/CDK9 kinase complex to specific promoters, through fusion to a DNA-binding domain of either Cyclin T1 or CDK9 kinase, stimulates transcription in vivo. Transcriptional enhancement was dependent on active CDK9, as a catalytically inactive form had no transcriptional effect. We determined that, unlike conventional activators, DNA-bound CDK9 does not activate enhancerless TATA-promoters unless TBP is overexpressed, suggesting that CDK9 acts in vivo at a step subsequent to TFIID recruitment DNA-bound. Finally, we determined that CDK9-mediated transcriptional activation is mediated by preferentially stimulating productive transcription elongation. PMID: 10467404 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Genes Cells. 1998 Aug;3(8):549-65. AMY-1, a novel C-MYC binding protein that stimulates transcription activity of C-MYC. Taira T, Maeda J, Onishi T, Kitaura H, Yoshida S, Kato H, Ikeda M, Tamai K, Iguchi-Ariga SM, Ariga H. Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12 Nishi 6, Kita-ku, Sapporo 060 Japan. BACKGROUND: The c-myc proto-oncogene has been suggested to play key roles in cell proliferation, differentiation, transformation and apoptosis. A variety of functions of C-MYC, the product of c-myc, are attributed to protein-protein interactions with various cellular factors including Max, YY1, p107, Bin1 and TBP. Max and YY1 bind to the C-terminal region of C-MYC, while p107, Bin1 and TBP bind to the N-terminal region covering myc boxes. The N-terminal region is involved in all the biological functions of C-MYC, and different proteins are therefore thought to interact with the N-terminal region of C-MYC to display different functions. RESULTS: We cloned two cDNAs which encode a novel C-MYC-binding protein of 11 kDa, designated AMY-1 (Associate of C-MYC). The two cDNAs, AMY-1L and AMY-1S, derived from alternative usage of polyadenylation signals, code for the same protein of 11 kDa. AMY-1 was bound via its C-terminal region to the N-terminal region of C-MYC (amino acids nos 58-148) corresponding to the transactivation domain. AMY-1 was localized in the cytoplasm in cells expressing c-myc at low levels, but in the nucleus in the cells of a high c-myc expression in transiently transfected cells. A similar difference in endogenous AMY-1 localization was observed during the cell cycle: AMY-1 translocated from cytoplasm to nucleus during the S phase when c-myc expression was increased. AMY-1 by itself did not recognize the E-box element, the MYC/Max binding sequence, nor did it transactivate via the element, but stimulated the activation of E-box-regulated transcription by MYC/Max. FISH analyses revealed that the amy-1 gene was located at 1p32.2-1p33 in human genome. CONCLUSIONS: AMY-1 is a 11 kDa protein which binds to the N-terminal region of C-MYC and stimulates the activation of E-box-dependent transcription by C-MYC. AMY-1, which mostly localizes in the cytoplasm, translocates into the nucleus in the S phase of the cell cycle upon an increase of c-myc expression, and may thus control the transcriptional activity of C-MYC. PMID: 9797456 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Oncogene. 1998 Sep 3;17(9):1149-57. Fli-1b is generated by usage of differential splicing and alternative promoter. Dhulipala PD, Lee L, Rao VN, Reddy ES. Department of Human Genetics, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102, USA. The proto-oncogene Fli-1, a member of Ets family is rearranged or activated through proviral integration in erythroleukemias, induced by Friends' Murine Leukemia Virus. The DNA binding domain (ETS domain) of Fli-1 is fused to the RNA binding domain of EWS by t(11q24:22q12) chromosomal translocation in Ewing's sarcoma and primitive neuroectodermal tumors. Screening of human cDNA libraries has identified two different 5'-termini and alternatively spliced forms of the human Fli-1 gene (Fli-1b), suggesting the possible existence of two independent promoters. The genomic sequence adjacent to the alternate exon of human Fli-1b gene shows functional promoter activity when cloned in promoter-less CAT expression vector and transfected into QT-6 cells. The transcription initiation (CAP) site and minimum promoter region necessary for function were localized. The 5'-flanking regions of human Fli-1b and mouse Fli-1 show 80% homology suggesting conserved promoter regulatory elements. The Fli-1b 5'-flanking sequence lacks canonical TATA or CCAAT boxes but contains a partially conserved TATA-like sequence at position 242. Several transcription factor binding sequences like ATF/CREB, E2A-PBX1, EBP, PEA-3, ETS-2, Sp-1, c-Myc, TBP, GATA-1 and Oct-3 were conserved in the promoter sequence. Functional promoter assays revealed that Fli-1b promoter shows very strong transcriptional activation compared to Fli-1 promoter. We also showed that variant Fli-1b has transcriptional activation properties similar to those of Fli-1. Fli-1b and Fli-1 show differential expression in various hematopoietic cell lines. This differential expression and promoter activities of Fli-1 and Fli-1b suggests that several mechanisms are involved in Fli-1 gene regulation which are mediated by many transcription factors. PMID: 9764825 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: J Biol Chem. 1998 Oct 9;273(41):26559-65. Tra1p is a component of the yeast Ada.Spt transcriptional regulatory complexes. Saleh A, Schieltz D, Ting N, McMahon SB, Litchfield DW, Yates JR 3rd, Lees-Miller SP, Cole MD, Brandl CJ. Department of Biochemistry, University of Western Ontario, London N6A 5C1, Canada. The yeast Ada and TBP class of Spt proteins interact in multiple complexes that are required for transcriptional regulation. We have identified Tra1p as a component of these complexes through tandem mass spectrometry analysis of proteins that associate with Ngg1p/Ada3p. TRA1 is an essential gene and encodes a 3744-amino acid protein that is a member of a group of proteins including the catalytic subunit of DNA-dependent protein kinase, ATM and TRRAP, with carboxyl-terminal regions related to phosphatidylinositol 3-kinases. The interaction between Tra1p and Ada/Spt components was verified by the reciprocal coimmunoprecipitation of Ada2p and Tra1p from whole cell extracts in one or more complexes containing Spt7p. Tra1p cofractionated with Ngg1p and Spt7p through consecutive chromatography on Mono Q, DNA-cellulose, and Superose 6 columns. Binding of Tra1p to DNA-cellulose required Ada components. The association of Tra1p with two Ada.Spt complexes was suggested by its cofractionation with Ngg1p and Spt7p in two peaks on the Mono Q column. In the absence of Ada2p, the elution profile of Tra1p shifted to a distinct peak. Despite the similarity of Tra1p to a group of putative protein kinases, we have not detected protein kinase activity within immunoprecipitates of Tra1p or the Ada.Spt complexes. PMID: 9756893 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Eur J Biochem. 1997 Aug 1;247(3):1166-73. Phosphorylation of human general transcription factors TATA-binding protein and transcription factor IIB by DNA-dependent protein kinase--synergistic stimulation of RNA polymerase II basal transcription in vitro. Chibazakura T, Watanabe F, Kitajima S, Tsukada K, Yasukochi Y, Teraoka H. Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, Japan. DNA-dependent protein kinase (DNA-PK) has been known to catalyze phosphorylation of a number of regulatory factors involved in DNA replication and transcription such as simian virus 40 T antigen, p53, c-Myc, Sp1, and RNA polymerase II (Pol II). We examined the possibility that DNA-PK phosphorylates the general transcription factors TATA-binding protein (TBP) and transcription factor (TF) IIB, which play key roles in the formation of transcription initiation complex with Pol II. By using a highly purified preparation of DNA-PK from Raji cells, both TBP and TFIIB were shown to be phosphorylated in vitro by DNA-PK. We then investigated the effect of the phosphorylation of these factors on Pol II basal transcription. Stepwise analysis of preinitiation complex formation by electrophoretic mobility shift assay revealed that the phosphorylation of TBP and TFIIB by DNA-PK did not affect the formation of promoter (P)-TBP and P-TBP-TFIIB complexes but synergistically stimulated the formation of P-TBP-TFIIB-TFIIF-Pol II complex. Similarly, combination of the phosphorylated TBP and TFIIB synergistically stimulated Pol II basal transcription from adenovirus major late promoter. These observations suggest that DNA-PK could positively regulate the Pol II basal transcription by phosphorylating TBP and TFIIB. PMID: 9288944 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Biochemistry. 1996 Jul 23;35(29):9584-93. Functional interaction of the c-Myc transactivation domain with the TATA binding protein: evidence for an induced fit model of transactivation domain folding. McEwan IJ, Dahlman-Wright K, Ford J, Wright AP. Department of Biosciences, Karolinska Institute, NOVUM, Huddinge, Sweden. iain.mcewan@cbt.ki.se c-Myc is a member of a family of sequence specific-DNA binding proteins that are thought to regulate the transcription of genes involved in normal cell growth, differentiation, and apoptosis. In order to understand how human c-myc functions as a transcription factor, we have studied the mechanism of action and structure of the N-terminal transactivation domain, amino acids 1-143. In a protein interaction assay, c-myc1-143 bound selectively to two basal transcription factors, the TATA binding protein (TBP) and the RAP74 subunit of TFIIF. Furthermore, the isolated c-myc transactivation domain competed for limiting factors required for the assembly of a functional preinitiation complex. This squelching of basal transcription was reversed in a dose-dependent manner by recombinant TBP. Taken together, these results identify TBP as an important target for the c-myc transactivation domain, during transcriptional initiation. Similar to other transactivation domains, the c-myc1-143 polypeptide showed little or no evidence of secondary structure, when measured by circular dichroism spectroscopy (CD) in aqueous solution. However, significant alpha-helical conformation was observed in the presence of the hydrophobic solvent trifluoroethanol. Strikingly, addition of TBP caused changes in the CD spectra consistent with induction of protein conformation in c-myc1-143 during interaction with the target factor. This change was specific for TBP as a similar effect was not observed in the presence of TFIIB. These data support a model in which target factors induce or stabilize a structural conformation in activator proteins during transcriptional transactivation. PMID: 8755740 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: 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] --------------------------------------------------------------- 25: J Biol Chem. 1996 May 31;271(22):13228-33. Invasion of the CAG triplet repeats by a complementary peptide nucleic acid inhibits transcription of the androgen receptor and TATA-binding protein genes and correlates with refolding of an active nucleosome containing a unique AR gene sequence. Boffa LC, Morris PL, Carpaneto EM, Louissaint M, Allfrey VG. Department of Experimental Oncology, Istituto Nazionale per la Ricerca, sul Cancro IST, Genoa 16132, Italy. The DNA sequence of the genes for the androgen receptor (AR) and TATA-binding protein (TBP), like many other genes encoding transcription factors, contains a series of tandem CAG repeats. Here we explore the capacity of complementary peptide nucleic acids (PNAs) to invade the CAG triplets of the AR and TBP genes in human prostatic cancer cells and show that the PNAs readily entered the nuclei of lysolecithin-permeabilized cells and effectively inhibited sense transcription of unique AR and TBP DNA sequences downstream of the site of PNA.DNA hybridization, but not upstream of that site. These PNAs had little or no effect on transcription of the c-myc gene, which lacks a CAG triplet domain. Conversely, a PNA complementary to a unique sequence of the c-myc gene did not inhibit transcription of the AR or TBP genes but did inhibit c-myc transcription. Comparisons of PNA effects on sense and antisense transcription of the AR, TBP, and c-myc genes confirm that progression of the RNA polymerase complex beyond the site of PNA.DNA hybridization is impaired in both directions. Suppression of the AR gene results in refolding of a transcriptionally active nucleosome containing a unique 17-mer AR DNA sequence. PMID: 8662737 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: Mol Cell Biol. 1996 May;16(5):2350-60. Heterogeneous nuclear ribonucleoprotein K is a transcription factor. Michelotti EF, Michelotti GA, Aronsohn AI, Levens D. Laboratory of Pathology, National Cancer Institute, National Institute of Health, Bethesda, Maryland 20892, USA. The CT element is a positively acting homopyrimidine tract upstream of the c-myc gene to which the well-characterized transcription factor Spl and heterogeneous nuclear ribonucleoprotein (hnRNP) K, a less well-characterized protein associated with hnRNP complexes, have previously been shown to bind. The present work demonstrates that both of these molecules contribute to CT element-activated transcription in vitro. The pyrimidine-rich strand of the CT element both bound to hnRNP K and competitively inhibited transcription in vitro, suggesting a role for hnRNP K in activating transcription through this single-stranded sequence. Direct addition of recombinant hnRNP K to reaction mixtures programmed with templates bearing single-stranded CT elements increased specific RNA synthesis. If hnRNP K is a transcription factor, then interactions with the RNA polymerase II transcription apparatus are predicted. Affinity columns charged with recombinant hnRNP K specifically bind a component(s) necessary for transcription activation. The depleted factors were biochemically complemented by a crude TFIID phosphocellulose fraction, indicating that hnRNP K might interact with the TATA-binding protein (TBP)-TBP-associated factor complex. Coimmunoprecipitation of a complex formed in vivo between hnRNP K and epitope-tagged TBP as well as binding in vitro between recombinant proteins demonstrated a protein-protein interaction between TBP and hnRNP K. Furthermore, when the two proteins were overexpressed in vivo, transcription from a CT element-dependent reporter was synergistically activated. These data indicate that hnRNP K binds to a specific cis element, interacts with the RNA polymerase II transcription machinery, and stimulates transcription and thus has all of the properties of a transcription factor. PMID: 8628302 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: 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] --------------------------------------------------------------- 28: EMBO J. 1995 Oct 16;14(20):5073-84. Programming of a repressed but committed chromatin structure during early development. Prioleau MN, Buckle RS, Mechali M. Institut Jacques Monod, Molecular Embryology Unit, Paris, France. The determination of chromatin for transcription during early development as well as the requirement for trans-acting factors during this period has been analysed in Xenopus. Basal transcription is repressed both during oogenesis and after the mid-blastula transition (MBT), and transactivators are required to relieve this repression. In contrast, transactivators cannot overcome the generalized transcriptional repression which occurs in embryos before MBT. However, they do bind to promoters leading to a repressed but preset chromatin structure. Experiments involving the pre-binding of TATA binding protein (TBP) or of the strong transactivator GAL4-VP16 further show that there is no limiting factor before the MBT, and that it is the recruitment and stabilization of the basal transcription machinery and not of transactivators which is repressed during early development. This multi-step process in gene activation, with activation of promoters temporally uncoupled from their commitment, may be of importance in the regulation of early embryonic events by providing molecular signposts for future determinations. PMID: 7588635 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Nature. 1995 Apr 27;374(6525):822-3. Stimulation of RNA polymerase II transcription initiation by recruitment of TBP in vivo. Klages N, Strubin M. Department of Genetics and Microbiology, University Medical Centre (CMU), Geneva, Switzerland. Eukaryotic transcriptional activators may stimulate RNA polymerase II activity by promoting assembly of preinitiation complexes on promoters through their interactions with one or more components of the basal machinery. On the basis of its central role in initiating transcription-complex formation upon binding to the TATA box, the general transcription factor TFIID, which includes the TATA-binding protein (TBP) and several TBP-associated factors, has been implicated as a target for activators. Consistent with this idea, an increasing number of activators have been reported to bind directly to TBP. To assess the functional importance of these in vitro interactions for transcriptional regulation in vivo, we made use of a novel strategy in yeast to show that a physical interaction with TBP is sufficient for a sequence-specific DNA-binding protein to increase initiation of transcription by RNA polymerase II. These results imply that binding of TFIID to promoter elements is a limiting step in transcription complex assembly in vivo. PMID: 7723829 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: Biochemistry. 1995 Mar 14;34(10):3438-45. The c-myc promoter binding protein (MBP-1) and TBP bind simultaneously in the minor groove of the c-myc P2 promoter. Chaudhary D, Miller DM. Department of Medicine, University of Alabama at Birmingham 35294-3300. The c-myc promoter binding protein (MBP-1) is a DNA binding protein which negatively regulates the expression of the human c-myc gene. MBP-1 binds to a sequence which overlaps the binding site for the general transcription factor TBP, within the c-myc P2 promoter region. Since TBP binds in the minor groove, MBP-1 might inhibit c-myc transcription by preventing the formation of a functional preinitiation complex. In support of this hypothesis, we have demonstrated that MPB-1 is a minor groove binding protein. In order to characterize MBP-1 binding, we substituted A-T base pairs in the MBP-1 binding site with I-C base pairs, which changes the major groove surface without altering the minor groove surface. This substitution did not inhibit the sequence-specific binding of MBP-1 and TBP. On the other hand, G-C to I-C substitution within the MBP-1 binding site alters the minor groove and prevents MBP-1 binding. Competitive electrophoretic mobility shift assays were used to show that berenil, distamycin, and mithramycin, all of which bind in the minor groove, compete with MBP-1 for binding to the MPB-1 binding site. These minor groove binding ligands also effectively inhibit the simultaneous DNA binding activity of both MBP-1 and TBP. We conclude that both MBP-1 and TBP can bind simultaneously in the minor groove of the TATA motif on the c-myc P2 promoter. This suggests that MBP-1 may negatively regulate c-myc gene expression by preventing efficient transcription initiation. PMID: 7880838 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: Cell. 1994 May 6;77(3):439-49. Competition between chromatin and transcription complex assembly regulates gene expression during early development. Prioleau MN, Huet J, Sentenac A, Mechali M. Institut Jacques Monod, Molecular Embryology Unit, Paris, France. Xenopus early development is characterized by a generalized absence of transcription, which resumes at the midblastula transition (MBT). We analyzed this regulation using a plasmid containing the c-myc promoter that is under the same developmental control when injected into fertilized eggs. We find that the repression of transcription can be relieved simply by preincubating the reporter plasmid with TATA binding protein (TBP). However, the repression of gene activity normally occurring before the MBT soon becomes dominant over this activation independent of cell cycle phases. This inactivation correlates with chromatin assembly, and titration of chromatin components not only relieves repression of TBP-dependent transcription but also permits the establishment of stable transcription during early development. Our data suggest that the large excess of histones represses gene activity during early development through a dynamic competition between chromatin assembly and transcription complex assembly. PMID: 8181062 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: Mol Cell Biol. 1994 Feb;14(2):1147-52. Intracellular association of the protein product of the c-myc oncogene with the TATA-binding protein. Maheswaran S, Lee H, Sonenshein GE. Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118. The c-myc proto-oncogene encodes nuclear phosphoproteins that bind DNA in a sequence-specific fashion and appear to function as transcriptional activators. Here we demonstrate that a 40-kDa nuclear protein coimmunoprecipitated with c-Myc specifically when nuclear proteins, extracted from nuclei of exponentially growing murine B-lymphoma WEHI 231 cells by using procedures for preparation of trans-acting factors, were reacted with anti-c-Myc antibodies made against different regions of the c-Myc protein. In contrast, preparation of nuclear lysates under denaturing conditions significantly reduced this coprecipitation. Upon incubation of WEHI 231 cells with the reversible chemical cross-linking agent dithiobis(succinimidyl propionate), the 40-kDa protein could be cross-linked to c-Myc protein intracellularly. Identification of the 40-kDa protein as the TATA-binding protein (TBP) of the TFIID transcription initiation complex was made by comigration and V-8 protease mapping, which yielded identical peptide fragments upon digestion of the 40-kDa protein and material immunoprecipitated with an anti-TBP specific antibody. Furthermore, in vitro-translated TBP bound to the amino-terminal portion of c-Myc. Column chromatography of cross-linked nuclear proteins showed TBP to be in a large-molecular-weight complex with c-Myc, consistent with a transcription initiation complex. These results indicate that intracellularly, c-Myc interacts with TBP, suggesting a mechanism of interaction of this oncoprotein with the basal transcription machinery. PMID: 8289795 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: Nature. 1993 Sep 23;365(6444):359-61. Direct role for Myc in transcription initiation mediated by interactions with TFII-I. Roy AL, Carruthers C, Gutjahr T, Roeder RG. Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021. The nuclear proto-oncoprotein Myc has been implicated in the control of cell proliferation and differentiation. Myc participates in transcription and belongs to the basic-helix-loop-helix (bHLH) family of regulatory proteins. Here we show that Myc interacts with TFII-I, a transcription initiation factor that activates core promoters through an initiator element (Inr). As previously observed for the bHLH activator USF, Myc was found to interact cooperatively with TFII-I at both Inr and upstream E-box promoter elements. However, in this case Myc interactions with TFII-I at the Inr lead to an inhibition of transcription initiation. This inhibition is selective for a TFII-I-dependent (as opposed to TFIIA-dependent) initiation pathway and correlates with the prevention of complex formation between the TATA-binding protein TBP (TFIID tau), TFII-I and the promoter. TBP probably interacts with Myc, but only slowly. These observations indicate that Myc has the potential to interact physically and functionally with components of the general transcription machinery. PMID: 8377829 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8489-93. TATA-binding protein and the retinoblastoma gene product bind to overlapping epitopes on c-Myc and adenovirus E1A protein. Hateboer G, Timmers HT, Rustgi AK, Billaud M, van 't Veer LJ, Bernards R. Division of Molecular Genetics, Massachusetts General Hospital, Charlestown. Using a protein binding assay, we show that the amino-terminal 204 amino acids of the c-Myc protein interact directly with a key component of the basal transcription factor TFIID, the TATA box-binding protein (TBP). Essentially the same region of the c-Myc protein also binds the product of the retinoblastoma gene, the RB protein. c-Myc protein coimmunoprecipitates with TBP in lysates of mammalian cells, demonstrating that the proteins are also complexed in vivo. A short peptide that spans the RB binding site of the E7 protein of human papilloma virus type 16 interferes with the binding of c-Myc to TBP. The same peptide also blocks binding of adenovirus E1A protein to TBP, suggesting that c-Myc and E1A bind to RB and TBP through overlapping epitopes. Furthermore, we show that binding of RB to E1A prevents association of E1A with TBP. Our data suggest that one of the functions of RB and RB-like proteins is to prevent interaction of viral and cellular oncoproteins, such as c-Myc and E1A, with TBP. PMID: 7690963 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: 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] ---------------------------------------------------------------