1: Hum Mol Genet. 2005 Sep 1;14(17):2501-9. Epub 2005 Jul 21. A dinucleotide deletion in the ankyrin promoter alters gene expression, transcription initiation and TFIID complex formation in hereditary spherocytosis. Gallagher PG, Nilson DG, Wong C, Weisbein JL, Garrett-Beal LJ, Eber SW, Bodine DM. Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06520-8064, USA. patrick.gallagher@yale.edu Ankyrin defects are the most common cause of hereditary spherocytosis (HS). In some HS patients, mutations in the ankyrin promoter have been hypothesized to lead to decreased ankyrin mRNA synthesis. The ankyrin erythroid promoter is a member of the most common class of mammalian promoters which lack conserved TATA, initiator or other promoter cis elements and have high G+C content, functional Sp1 binding sites and multiple transcription initiation sites. We identified a novel ankyrin gene promoter mutation, a TG deletion adjacent to a transcription initiation site, in a patient with ankyrin-linked HS and analyzed its effects on ankyrin expression. In vitro, the mutant promoter directed decreased levels of gene expression, altered transcription initiation site utilization and exhibited defective binding of TATA-binding protein (TBP) and TFIID complex formation. In a transgenic mouse model, the mutant ankyrin promoter led to abnormalities in gene expression, including decreased expression of a reporter gene and altered transcription initiation site utilization. These data indicate that the mutation alters ankyrin gene transcription and contributes to the HS phenotype by decreasing ankyrin gene synthesis via disruption of TFIID complex interactions with the ankyrin core promoter. These studies support the model that in promoters that lack conserved cis elements, the TFIID complex directs preinitiation complex formation at specific sites in core promoter DNA and provide the first evidence that disruption of TBP binding and TFIID complex formation in this type of promoter leads to alterations in start site utilization, decreased gene expression and a disease phenotype in vivo. PMID: 16037067 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: EMBO J. 2005 Aug 3;24(15):2753-67. Epub 2005 Jul 14. TAF4 inactivation in embryonic fibroblasts activates TGF beta signalling and autocrine growth. Mengus G, Fadloun A, Kobi D, Thibault C, Perletti L, Michel I, Davidson I. Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, Illkirch, France. We have inactivated transcription factor TFIID subunit TBP-associated factor 4 (TAF4) in mouse embryonic fibroblasts. Mutant taf4(-/-) cells are viable and contain intact TFIID comprising the related TAF4b showing that TAF4 is not an essential protein. TAF4 inactivation deregulates more than 1000 genes indicating that TFIID complexes containing TAF4 and TAF4b have distinct target gene specificities. However, taf4(-/-) cell lines have altered morphology and exhibit serum-independent autocrine growth correlated with the induced expression of several secreted mitotic factors and activators of the transforming growth factor beta signalling pathway. In addition to TAF4 inactivation, many of these genes can also be induced by overexpression of TAF4b. A competitive equilibrium between TAF4 and TAF4b therefore regulates expression of genes controlling cell proliferation. We have further identified a set of genes that are regulated both by TAF4 and upon adaptation to serum starvation and which may be important downstream mediators of serum-independent growth. Our study also shows that TAF4 is an essential cofactor for activation by the retinoic acid receptor and CREB, but not for Sp1 and the vitamin D3 receptor. PMID: 16015375 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Yeast. 2005 May;22(7):511-21. Mammalian transcription activation domains of VP16, AP2 and CTF activate transcription in a whole cell extract from Schizosaccharomyces pombe through the SRB/mediator. Tamayo E, Bernal G, Maldonado E. Programa de Biologia Celular y Molecular, Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile. The acidic-rich activation domain of VP16 and the proline-rich activation domains of human AP2 and human CTF are able to activate transcription in a whole cell extract from Schizosaccharomyces pombe, whereas the glutamine-rich domains of Sp1 and Oct2 are unable to activate transcription in this system. Immunodepletion experiments of the whole cell extracts using specific antibodies against pombe TAF110, pombe TAF 72, pombe TBP and Srb4 shows that the activation of transcription by VP16, AP2 and CTF is through the mediator, since depletion of Srb4 inhibits activated transcription but does not inhibit basal transcription. Immunodepletion of TBP causes inhibition of both activated and basal transcription. On the other hand, immunodepletion of TAFs does not have an effect on either activated or basal transcription. Purified RNA polymerase holoenzyme is able to rescue the transcriptional activation activity of the anti-Srb4 immunodepleted extract. Moreover, we demonstrate that the mediator is needed for basal transcription of a TATA-less promoter. PMID: 15942925 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Dev Dyn. 2005 Apr;232(4):1021-30. Genomic, cDNA, and embryonic expression analysis of zebrafish transforming growth factor beta 3 (tgfbeta3). Cheah FS, Jabs EW, Chong SS. Department of Pediatrics, National University of Singapore and Hospital, Singapore 119074, Singapore. TGFbeta3, a member of the transforming growth factor beta family, regulates a spectrum of biological processes and is involved in mammalian pulmonary and craniofacial development. Homologs of human TGFbeta3 have been identified in several vertebrate species. We sequenced a cDNA clone of zebrafish tgfbeta3, consisting of a 271-bp 5' untranslated region, a 1,233-bp open reading frame that encodes a predicted 410 amino acid peptide, and a 527-bp 3' untranslated region. Using 5' rapid amplification of cDNA ends, the transcription start site of this gene was determined to lie an additional 29 nucleotides upstream. The gene is composed of seven exons and maps to a segment of linkage group 17 that is syntenic to the human TGFbeta3 locus on chromosome 14q24. One stimulating protein 1 (Sp1) and two (TATA binding protein) (TBP) transcription factor binding sites were identified in the putative promoter segment upstream of the transcription start site. Comparative alignment analysis revealed a high degree of tgfbeta3 nucleotide and amino acid identity between zebrafish and other species, including complete conservation of the cysteine knot structure that facilitates protein-protein interaction. Also, 9 of 10 amino acid residues critical for ligand/receptor binding in human TGFbeta3 are conserved in zebrafish, suggesting a high degree of functional conservation even in lower vertebrates. Zebrafish tgfbeta3 transcripts were first detected in the notochord (10 somite to high-pec stage), followed by expression in the developing pharyngeal arch and neurocranial cartilage (18 somite to protruding mouth stage), lens and heart (21 somite to protruding mouth stage), and pectoral fins (prim-25 to protruding mouth stage). The strong expression in the pectoral fins, not reported in the orthologous mammalian forelimb, suggests a modified or novel function of tgfbeta3 during early fish development. Copyright 2005 Wiley-Liss, Inc. PMID: 15739231 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: BMC Evol Biol. 2005 Feb 13;5(1):15. The architecture of mammalian ribosomal protein promoters. Perry RP. Fox Chase Cancer Center Philadelphia, PA 19111, USA. RP_Perry@fccc.edu BACKGROUND: Mammalian ribosomes contain 79 different proteins encoded by widely scattered single copy genes. Coordinate expression of these genes at transcriptional and post-transcriptional levels is required to ensure a roughly equimolar accumulation of ribosomal proteins. To date, detailed studies of only a very few ribosomal protein (rp) promoters have been made. To elucidate the general features of rp promoter architecture, I made a detailed sequence comparison of the promoter regions of the entire set of orthologous human and mouse rp genes. RESULTS: A striking evolutionarily conserved feature of most rp genes is the separation by an intron of the sequences involved in transcriptional and translational regulation from the sequences with protein encoding function. Another conserved feature is the polypyrimidine initiator, which conforms to the consensus (Y)2C+1TY(T)2(Y)3. At least 60 % of the rp promoters contain a largely conserved TATA box or A/T-rich motif, which should theoretically have TBP-binding capability. A remarkably high proportion of the promoters contain conserved binding sites for transcription factors that were previously implicated in rp gene expression, namely upstream GABP and Sp1 sites and downstream YY1 sites. Over 80 % of human and mouse rp genes contain a transposable element residue within 900 bp of 5' flanking sequence; very little sequence identity between human and mouse orthologues was evident more than 200 bp upstream of the transcriptional start point. CONCLUSIONS: This analysis has provided some valuable insights into the general architecture of mammalian rp promoters and has identified parameters that might coordinately regulate the transcriptional activity of certain subsets of rp genes. PMID: 15707503 [PubMed - in process] --------------------------------------------------------------- 6: Genome Res. 2004 Aug;14(8):1562-74. Epub 2004 Jul 15. Clustering of DNA sequences in human promoters. FitzGerald PC, Shlyakhtenko A, Mir AA, Vinson C. Genome Analysis Unit, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. We have determined the distribution of each of the 65,536 DNA sequences that are eight bases long (8-mer) in a set of 13,010 human genomic promoter sequences aligned relative to the putative transcription start site (TSS). A limited number of 8-mers have peaks in their distribution (cluster), and most cluster within 100 bp of the TSS. The 156 DNA sequences exhibiting the greatest statistically significant clustering near the TSS can be placed into nine groups of related sequences. Each group is defined by a consensus sequence, and seven of these consensus sequences are known binding sites for the transcription factors (TFs) SP1, NF-Y, ETS, CREB, TBP, USF, and NRF-1. One sequence, which we named Clus1, is not a known TF binding site. The ninth sequence group is composed of the strand-specific Kozak sequence that clusters downstream of the TSS. An examination of the co-occurrence of these TF consensus sequences indicates a positive correlation for most of them except for sequences bound by TBP (the TATA box). Human mRNA expression data from 29 tissues indicate that the ETS, NRF-1, and Clus1 sequences that cluster are predominantly found in the promoters of housekeeping genes (e.g., ribosomal genes). In contrast, TATA is more abundant in the promoters of tissue-specific genes. This analysis identified eight DNA sequences in 5082 promoters that we suggest are important for regulating gene expression. Copyright 2004 Cold Spring Harbor Laboratory Press ISSN PMID: 15256515 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: J Biochem (Tokyo). 2004 Jun;135(6):683-93. Bombyx Y-box protein BYB facilitates specific DNA interaction of various DNA binding proteins independently of the cold shock domain. Takiya S, Nishita Y, Ishikawa S, Ohno K, Tamura TA, Suzuki Y. Laboratory of Gene Function and Regulation, CAST, Hokkaido University, North 10, West 8, Kita-ku, Sapporo 060-0810, Japan. takiya@rmg.hokudai.ac.jp A new member of the Y-box protein family of the silkworm Bombyx mori (BYB) was co-purified with the fibroin gene enhancer-binding protein FMBP-1, and stimulated the binding of FMBP-1 to its cognate DNA element. However, the stimulatory effect was not specific to FMBP-1, BYB also enhancing the binding of mammalian transcription factors OTF2, SP1 and AP2 to their specific binding elements. Besides the above transcription regulatory factors, BYB facilitated the binding of basal transcription factor TBP, and enhanced transcription from the adenovirus 2 major late promoter in a reconstituted transcription system. Moreover, BYB stimulated the reactions of some restriction endonucleases under cold conditions. The C-terminal region of BYB was sufficient for these stimulatory effects, and the highly conserved cold shock domain (CSD) in the N-terminal region was dispensable. GST-pull down experiments showed that the C-terminal region could interact with DNA independently of the CSD. The above results suggest that the C-terminal region of BYB causes the active interaction of various DNA binding proteins with their targets. Such a function of the C-terminal region of BYB may partly explain the functional diversity of Y-box proteins. PMID: 15213243 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: 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] --------------------------------------------------------------- 9: Nucleic Acids Res. 2003 Aug 1;31(15):4523-30. Low pH enhances Sp1 DNA binding activity and interaction with TBP. Torigoe T, Izumi H, Yoshida Y, Ishiguchi H, Okamoto T, Itoh H, Kohno K. Department of Molecular Biology, University of Occupational and Environmental Health, School of Medicine, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan. Sp1 is involved in the regulation of a wide variety of genes, including housekeeping genes and genes involved in tumor growth. Sp1 is a member of the C2-H2 zinc-finger family and is important for protection against cellular acidosis in cells that grow under hypoxic or acidic conditions, such as tumor cells. To obtain an insight into the molecular mechanisms underlying pH-dependent transcription by Sp1, both its DNA binding activity and its interaction with TATA binding protein (TBP) were investigated under various pH conditions. We show here that the DNA binding activity of Sp1 increased and Sp1 formed a stable interaction with TBP at low pH. These findings indicate that pH changes significantly modulate the activity of Sp1 and thus contribute to the cellular response under hypoxic or acidic conditions. PMID: 12888513 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: J Biol Chem. 2003 Sep 26;278(39):38068-75. Epub 2003 Jul 10. Interaction between the varicella zoster virus IE62 major transactivator and cellular transcription factor Sp1. Peng H, He H, Hay J, Ruyechan WT. Department of Microbiology, University at Buffalo, Buffalo, New York 14214, USA. The varicella zoster virus (VZV) IE62 protein is involved in the activation of expression of all three kinetic classes of VZV proteins. Analysis of the viral promoter for VZV glycoprotein I has shown that the cellular factor Sp1 is involved in or required for the observed IE62 mediated activation. Co-immunoprecipitation experiments show that the two proteins are present in a complex in VZV-infected cells. Protein affinity pull-down assays using recombinant proteins showed that IE62 and Sp1 interact in the absence of any other viral and cellular proteins. Mapping studies using GST-fusion proteins containing truncations of IE62 and Sp1 have delimited the interacting regions to amino acids 612-778 in Sp1 and amino acids 226-299 in IE62. The region identified in Sp1 is involved in DNA-binding, synergistic Sp1 activation, and Sp1 interaction with cellular transcription factors. The interacting region identified in IE62 overlaps with or borders on sites involved in interactions with the VZV IE4 protein and the cellular factors TBP and TFIIB. Assays using wild-type and mutant promoter elements indicate that Sp1 is involved in recruitment of IE62 to the gI promoter and IE62 enhances Sp1 and TBP binding. PMID: 12855699 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: J Biol Chem. 2003 Apr 11;278(15):12992-3002. Epub 2003 Feb 4. Transcription factor IID recruitment and Sp1 activation. Dual function of TAF1 in cyclin D1 transcription. Hilton TL, Wang EH. University of Washington, Department of Pharmacology, School of Medicine, Health Sciences Center, Box 357280, Seattle, Washington 98195-7280, USA. Cyclin D1 is an oncogene that regulates progression through the G(1) phase of the cell cycle. A temperature-sensitive missense mutation in the transcription factor TAF1/TAF(II)250 induces the mutant ts13 cells to arrest in late G(1) by decreasing transcription of cell cycle regulators, including cyclin D1. Here we provide evidence that TAF1 serves two independent functions, one at the core promoter and one at the upstream activating Sp1 sites of the cyclin D1 gene. Using in vivo genomic footprinting, we have identified protein-DNA interactions within the cyclin D1 core promoter that are disrupted upon inactivation of TAF1 in ts13 cells. This 33-bp segment, which we termed the TAF1-dependent element 1 (TDE1), contains an initiation site that displays homology to the consensus motif and is sufficient to confer a requirement for TAF1 function. Electrophoretic mobility shift assays reveal that binding of ts13-TAF1-containing TFIID complexes to the cyclin D1 TDE1 occurs at 25 degrees C but not at 37 degrees C in vitro and involves the initiator element. Temperature-dependent DNA binding activity is also observed for TAF1-TAF2 heterodimers assembled with the ts13 mutant but not the wild-type TAF1 protein. These data suggest that a function of TAF is required for the interaction of TFIID with the cyclin D1 initiator. Our finding that recruitment of TFIID, by insertion of a TBP binding site upstream of the TDE1, restores basal but not activated transcription supports the model that TAF1 carries out two independent functions at the cyclin D1 promoter. PMID: 12569092 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Hum Mol Genet. 2002 Apr 15;11(8):905-14. Huntingtin inclusions do not deplete polyglutamine-containing transcription factors in HD mice. Yu ZX, Li SH, Nguyen HP, Li XJ. Department of Human Genetics, Emory University, School of Medicine, Atlanta, GA 30322, USA. A pathological hallmark of polyglutamine diseases is the presence of inclusions or aggregates of the expanded polyglutamine protein. Polyglutamine inclusions are present in the neuronal nucleus in a number of inherited neurodegenerative disorders, including Huntington disease (HD). Recent studies suggest that polyglutamine inclusions may sequester polyglutamine-containing transcription factors and deplete their concentration in the nucleus, leading to altered gene expression. To test this hypothesis, we examined the expression and localization of the polyglutamine-containing or glutamine-rich transcription factors TBP, CBP and Sp1 in HD mouse models. All three transcription factors were diffusely distributed in the nucleus, despite the presence of abundant intranuclear inclusions. There were no differences in the nuclear staining of these transcription factors between HD and wild-type mouse brains. Although some CBP staining appeared as dots in the selective brain regions (e.g. hypothalamus and amygdala), double labeling showed that most CBP was not co-localized with huntingtin nuclear inclusions. Electron microscopy confirmed that CBP was diffusely distributed in the nucleus. Western blots showed that these transcription factors were not trapped in huntingtin inclusions. In the striatum of HD mice, which suffers a significant reduction in the expression of a number of genes, mutant huntingtin was present in both an aggregated and a diffuse form. These findings suggest that altered gene expression may result from the interactions of soluble mutant huntingtin with nuclear transcription factors, rather than from the depletion of transcription factors by nuclear inclusions. PMID: 11971872 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: J Biol Chem. 2002 Jul 19;277(29):25877-83. Epub 2002 Apr 9. The rod cGMP-phosphodiesterase beta-subunit promoter is a specific target for Sp4 and is not activated by other Sp proteins or CRX. Lerner LE, Gribanova YE, Whitaker L, Knox BE, Farber DB. Jules Stein Eye Institute, Department of Ophthalmology, UCLA School of Medicine, Los Angeles, California 90095, USA. The beta-subunit of cGMP-phosphodiesterase (beta-PDE) is a key protein in phototransduction expressed exclusively in rod photoreceptors. It is necessary for visual function and for structural integrity of the retina. beta-PDE promoter deletions showed that the -45/-23 region containing a consensus Crx-response element (CRE) was necessary for low level transcriptional activity. Overexpressed Crx modestly transactivated this promoter in 293 human embryonic kidney cells; however, mutation of CRE had no significant effect on transcription either in transfected Y79 retinoblastoma cells or Xenopus embryonic heads. Thus, Crx is unlikely to be a critical beta-PDE transcriptional regulator in vivo. Interestingly, although the beta/GC element (-59/-49) binds multiple Sp transcription factors in vitro, only Sp4, but not Sp1 or Sp3, significantly enhanced beta-PDE promoter activity. Thus, the Sp4-mediated differential activation of the beta-PDE transcription defines the first specific Sp4 target gene reported to date and implies the importance of Sp4 for retinal function. Further extensive mutagenesis of the beta-PDE upstream sequences showed no additional regulatory elements. Although this promoter lacks a canonical TATA box or Inr element, it has the (T/A)-rich beta/TA sequence located within the -45/-23 region. We found that it binds purified TBP and TFIIB in gel mobility shift assays with cooperative enhancement of binding affinity. PMID: 11943774 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: J Biol Chem. 2002 Jan 25;277(4):2485-97. Epub 2001 Nov 5. Cooperative coactivation of estrogen receptor alpha in ZR-75 human breast cancer cells by SNURF and TATA-binding protein. Saville B, Poukka H, Wormke M, Janne OA, Palvimo JJ, Stoner M, Samudio I, Safe S. Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA. SNURF is a small RING finger protein that binds the zinc finger region of steroid hormone receptors and enhances Sp1- and androgen receptor-mediated transcription in COS and CV-1 cells. In this study, we show that SNURF coactivates both wild-type estrogen receptor alpha (ERalpha) (4-fold)- and HE19 (ERalpha deletion of activation function 1 (AF1)) (210-fold)-mediated activation of an estrogen-responsive element promoter in ZR-75 cells. In mammalian two-hybrid assays in ZR-75 cells SNURF interactions were estrogen (E2)-dependent and were not observed with the antiestrogen ICI 182,780. ERalpha interacted with multiple regions of SNURF; SNURF interactions with ERalpha were dependent on AF2, and D538N, E542Q, and D545N mutations in helix 12 abrogated both SNURF-ERalpha binding and coactivation. Moreover, peptide fusion proteins that inhibit interactions between helix 12 of ERalpha with LXXLL box-containing proteins also blocked ERalpha coactivation by SNURF. However, cotransfection of SNURF with prototypical steroid receptor coactivators 1, 2, and 3 that contain LXXLL box motifs did not enhance E2 responsiveness, whereas TATA-binding protein (TBP) and SNURF cooperatively coactivated ERalpha-mediated transactivation. The results are consistent with a unique model for cooperative coactivation of ERalpha that requires ligand binding, repositioning of helix 12, recruitment of TBP, and interaction with SNURF, which binds both ERalpha and TBP. PMID: 11696545 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Neuropathology. 2000 Dec;20(4):319-25. Pathology of CAG repeat diseases. Yamada M, Tsuji S, Takahashi H. Department of Pathology, Brain Research Institute, Niigata University, Japan. nori@bri.niigata-u.ac.jp Neuronal intranuclear inclusions have become the neuropathological signature of the CAG repeat diseases, although their cytotoxicity is a matter of controversy. It has been demonstrated that the inclusions in dentatorubral-pallidoluysian atrophy (DRPLA) and Machado-Joseph disease (MJD) were immunopositive for several transcription factors such as TATA-binding protein (TBP), TBP-associated factor (TAF(II)130), Sp1, cAMP-responsive element-binding protein (CREB) and CREB-binding protein, suggesting that neuronal degeneration in polyglutamine diseases may result from nuclear depletion of transcription factors containing the glutamine-rich domain. It was also revealed that, in the DRPLA brain, expanded polyglutamine stretches were diffusely accumulated in neuronal nucleoplasm. This nuclear pathology involved many neurons in various nervous system regions, such as the cerebral cortex, thalamus, substantia nigra, pontine nuclei, reticular formation and inferior olive, in addition to the previously recognized affected regions. The diffuse nuclear labeling was also detected in MJD, Huntington's disease, and spinal and bulbar muscular atrophy, suggesting that this nuclear pathology may be a characteristic feature and may exert certain influence on certain nuclear functions of many neurons in the CAG repeat diseases. Publication Types: Review Review, Tutorial PMID: 11211058 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Oncogene. 2000 Aug 3;19(33):3717-26. Activation of the insulin-like growth factor II transcription by aflatoxin B1 induced p53 mutant 249 is caused by activation of transcription complexes; implications for a gain-of-function during the formation of hepatocellular carcinoma. Lee YI, Lee S, Das GC, Park US, Park SM, Lee YI. Bioscience Research Division, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon. Aflatoxin B1 (AFB1) induced mutation of the p53 gene at codon 249 (p53mt249) is critical during the formation of hepatocellular carcinoma (HCC) following hepatitis B virus (HBV) infection. p53mt249 markedly increases insulin-like growth factor II (IGF-II) transcription largely from promoter 4, accumulating the fetal form of IGF-II. Modulation of the transcription factor binding to IGF-II P4 by wild-type p53 and p53mt249 was identified. Wild-type p53 inhibited binding of transcription factors Sp1 and TBP on the P4 promoter, while p53mt249 enhanced the formation of transcriptional complexes through enhanced DNA-protein (Sp1 or TBP) and protein-protein (Sp1 and TBP) interactions. p53mt249 stimulates transcription factor Sp1 phosphorylation which might be a cause of increased transcription factor binding on the P4 promoter while wild-type p53 does not. Transfection of hepatocytes with p53mt249 impaired induction of apoptosis by the HBV-X protein and TNF-alpha. Therefore, the blocking of apoptosis through enhanced production of IGF-II should provide a favorable opportunity for the selection of transformed hepatocytes. These results explain the molecular basis for the genesis of HCC by p53mt249 which was found to be induced by a potent mutagen, AFB1. PMID: 10949925 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: J Biol Chem. 2000 Sep 22;275(38):29847-56. Assembly of partial TFIID complexes in mammalian cells reveals distinct activities associated with individual TATA box-binding protein-associated factors. Furukawa T, Tanese N. Department of Microbiology, New York University School of Medicine, New York, New York 10016, USA. The TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s) compose the general transcription factor TFIID. The TAF(II) subunits mediate activated transcription by RNA polymerase II by interacting directly with site-specific transcriptional regulators. TAF(II)s also participate in promoter recognition by contacting core promoter elements in the context of TFIID. To further dissect the contribution of individual TAF(II) subunits to mammalian TFIID function, we employed a vaccinia virus-based protein expression system to study protein-protein interactions and complex assembly. We identified the domains of human (h) TAF(II)130 required for TAF(II)-TAF(II) interactions and formation of a complex with hTBP, hTAF(II)100, and hTAF(II)250. Functional analysis of partial TFIID complexes formed in vivo indicated that hTAF(II)130 was required for transcriptional activation by Sp1 in vitro. DNase I footprinting experiments demonstrated that purified hTBP/hTAF(II)250 complex reconstituted with or without additional TAF(II)s was significantly reduced for TATA box binding (as much as 9-fold) compared with free hTBP. By contrast, hTAF(II)130 stabilized binding of hTBP to the TATA box, whereas hTAF(II)100 had little effect. Thus, our biochemical analysis supports the notion that TAF(II)s possess distinct functions to regulate the activity of TFIID. PMID: 10896937 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Mol Cell Biol. 2000 Jun;20(12):4350-8. Artificial recruitment of TFIID, but not RNA polymerase II holoenzyme, activates transcription in mammalian cells. Dorris DR, Struhl K. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA. In yeast cells, transcriptional activation occurs when the RNA polymerase II (Pol II) machinery is artificially recruited to a promoter by fusing individual components of this machinery to a DNA-binding domain. Here, we show that artificial recruitment of components of the TFIID complex can activate transcription in mammalian cells. Surprisingly, artificial recruitment of TATA-binding protein (TBP) activates transiently transfected and chromosomally integrated promoters with equal efficiency, whereas artificial recruitment of TBP-associated factors activates only chromosomal reporters. In contrast, artificial recruitment of various components of the mammalian Pol II holoenzyme does not confer transcriptional activation, nor does it result in synergistic activation in combination with natural activation domains. In the one case examined in more detail, the Srb7 fusion failed to activate despite being associated with the Pol II holoenzyme and being directly recruited to the promoter. Interestingly, some acidic activation domains are less effective when the promoter is chromosomally integrated rather than transiently transfected, whereas the Sp1 glutamine-rich activation domain is more effective on integrated reporters. Thus, yeast and mammalian cells differ with respect to transcriptional activation by artificial recruitment of the Pol II holoenzyme. PMID: 10825198 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Biochem Biophys Res Commun. 2000 Mar 16;269(2):302-8. Artificial recruitment of Sp1 or TBP can replace the role of IE1 in the synergistic transactivation by IE1 and IE2. Kim JM, Hong Y, Kim S. Institute for Molecular Biology and Genetics, Seoul National University, Seoul, 151-742, Korea. The IE1 and IE2 proteins of human cytomegalovirus transactivate various viral and cellular promoters in a synergistic manner, but the mechanism of their action has not been well elucidated. Here we have examined the IE1-IE2 synergy by artificial recruitment of either Sp1 or TBP to the promoter. We found that in the presence of Sp1, the synergistic effect of IE1 on IE2-mediated transactivation dramatically decreased. Furthermore, a 117-amino acids glutamine-rich fragment of Sp1, which can interact with dTAF(II)110 and hTAF(II)130, was sufficient to replace the role of IE1 in IE1-IE2 synergism. It was also found that TBP recruitment to the promoter markedly decreased the synergistic effect of IE1 on IE2-mediated transactivation. These results suggested that in the context of the synergism between IE1 and IE2, the function of IE1 might overlap with that of Sp1, for example by recruiting the TFIID complex. Copyright 2000 Academic Press. PMID: 10708547 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: J Mol Cell Cardiol. 2000 Jan;32(1):95-108. Identification of cis-acting DNA elements required for expression of the human cardiac troponin I gene promoter. Bhavsar PK, Dellow KA, Yacoub MH, Brand NJ, Barton PJ. National Heart and Lung Institute, Imperial College School of Medicine, Dovehouse Street, London, SW3 6LY, UK. The human cardiac troponin I (TnIc) gene exhibits both cardiac-specific and developmentally regulated expression. The structure and expression of this gene as well as the identification of putative regulatory elements have been described previously. This study shows that a minimal promoter containing 98 bp of sequence is sufficient to drive transcription in neonatal rat cardiac myocytes. This region contains several putative cis -regulatory elements including an Initiator element surrounding the start site of transcription, an A/T-rich (TATA/MEF-2) element, two GATA elements and a cytosine-rich region containing overlapping CACC box and Sp1 elements. Using electrophoretic mobility shift assays (EMSAs) this study demonstrates the binding of MEF-2, Oct-1, and recombinant TBP to the A/T-rich element and of GATA-4 to both GATA elements. The CACC/Sp element binds the zinc finger transcription factors Sp1 and Sp3 in addition to an unidentified complex present in neonatal rat cardiac myocytes. Mutation of each of these sites has a deleterious effect on promoter activity as assayed by transient transfection into cardiac myocytes. The data suggest that transcriptional activity of the human TnIc gene can be driven by a compact promoter region and that within this region GATA, MEF-2 Sp1 and CACC box-binding factors are required for optimal activity. Furthermore, a comparison with data obtained for identical elements in the promoters of rodent TnIc genes identifies differences between species which may be of consequence for species-specific promoter function. Copyright 2000 Academic Press. PMID: 10652194 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: J Gen Virol. 2000 Jan;81(Pt 1):37-46. Transactivation activity of the human cytomegalovirus IE2 protein occurs at steps subsequent to TATA box-binding protein recruitment. Kim JM, Hong Y, Jeang KT, Kim S. Institute for Molecular Biology and Genetics, Seoul National University, Building 105, Kwan-Ak-Gu, Seoul 151-742, Korea. The IE2 protein of human cytomegalovirus transactivates viral and cellular promoters through a wide variety of cis-elements, but the mechanism of its action has not been well characterized. Here, IE2-Sp1 synergy and IE2-TATA box-binding protein (TBP) interaction are examined by artificial recruitment of either Sp1 or TBP to the promoter. It was found that IE2 could cooperate with DNA-bound Sp1. A 117 amino acid glutamine-rich fragment of Sp1, which can interact with Drosophila TAF(II)110 and human TAF(II)130, was sufficient for the augmentation of IE2-driven transactivation. In binding assays in vitro, IE2 interacted directly with the C-terminal region of Sp1, which contains the zinc finger DNA-binding domain, but not with its transactivation domain, suggesting that synergy between IE2 and the transactivation domain of Sp1 might be mediated by other proteins such as TAF or TBP. It was also found that TBP recruitment to the promoter markedly increased IE2-mediated transactivation. Thus, IE2 acts synergistically with DNA-bound Sp1 and DNA-bound TBP. These results suggest that, in human cytomegalovirus IE2 transactivation, Sp1 functions at an early step such as recruitment of TBP and IE2 acts to accelerate rate-limiting steps after TBP recruitment. PMID: 10640540 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: 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] --------------------------------------------------------------- 23: EMBO J. 1999 Sep 1;18(17):4835-45. DNA binding site selection by RNA polymerase II TAFs: a TAF(II)250-TAF(II)150 complex recognizes the initiator. Chalkley GE, Verrijzer CP. Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. Basal transcription factor TFIID comprises the TATA-box-binding protein, TBP, and associated factors, the TAF(II)s. Previous studies have implicated TAF(II)250 and TAF(II)150 in core promoter selectivity of RNA polymerase II. Here, we have used a random DNA binding site selection procedure to identify target sequences for these TAFs. Individually, neither TAF(II)250 nor TAF(II)150 singles out a clearly constrained DNA sequence. However, a TAF(II)250-TAF(II)150 complex selects sequences that match the Initiator (Inr) consensus. When in a trimeric complex with TBP, these TAFs select Inr sequences at the appropriate distance from the TATA-box. Point mutations that inhibit binding of the TAF(II)250-TAF(II)150 complex also impair Inr function in reconstituted basal transcription reactions, underscoring the functional relevance of Inr recognition by TAFs. Surprisingly, the precise DNA sequence at the start site of transcription influences transcriptional regulation by the upstream activator Sp1. Finally, we found that TAF(II)150 specifically binds to four-way junction DNA, suggesting that promoter binding by TFIID may involve recognition of DNA structure as well as primary sequence. Taken together, our results establish that TAF(II)250 and TAF(II)150 bind the Inr directly and that Inr recognition can determine the responsiveness of a promoter to an activator. PMID: 10469661 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Proc Natl Acad Sci U S A. 1999 Aug 31;96(18):10092-7. In vivo transcription factor recruitment during thyroid hormone receptor-mediated activation. Kim MK, Lee JS, Chung JH. Molecular and Cellular Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1654, USA. Thyroid hormone receptor (TR) can act as both a transcriptional activator and a silencer. Optimal activation by TR requires synergism with activator(s) bound to the promoter (promoter proximal activator). It is thought that liganded TR either helps to recruit preinitiation complexes (PIC) to the promoter or activates the PIC already recruited. However, the studies analyzing the TR action on the PIC formation were done in vitro and, therefore, it is not clear how relevant they are to the in vivo TR action. For example, in vivo, the TR can act from distances equal to or greater than a kilobase from the promoter, but such distant effect is not reproducible in vitro. In this study, we used the PIN*POINT (ProteIN POsition Identification with Nuclease Tail) assay to define the molecular mechanism of TR action on transcription from the thymidine kinase promoter in the cellular context. We demonstrate that the recruitment of promoter-proximal activator Sp1, and the components of the basal transcription factors such as TBP, TFIIB, and Cdk7, is enhanced with thyroid hormone activation. Our results suggest that DNA forms a loop with TR-mediated activation to accommodate interactions between the liganded TR complex and the complex formed on the promoter. We also show that Sp1 bound to the promoter is essential for the DNA looping and recruitment of basal transcription factors such as TFIIB and Cdk7 but not for recruitment of TBP. On the basis of these findings, we present a model that illustrates the molecular mechanism of TR-mediated activation in vivo. PMID: 10468567 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Gene. 1999 Aug 20;236(2):197-208. Unlocking the mechanisms of transcription factor YY1: are chromatin modifying enzymes the key? Thomas MJ, Seto E. Molecular Oncology Program, H. Lee Moffitt Cancer Center & Research Institute, University of South Florida, Tampa, FL 33612, USA. The transcription factor YY1 is a complex protein that is involved in repressing and activating a diverse number of promoters. Numerous studies have attempted to understand how this one factor can act both as a repressor and an activator in such a wide set of different contexts. The fact that YY1 interacts with a number of key regulatory proteins (e.g. TBP, TFIIB, TAFII55, Sp1, and E1A) has suggested that these interactions are important for determining which particular function of YY1 is displayed at a specific promoter. Two groups of proteins, previously known to function as corepressors and coactivators, that now seem likely to modulate YY1's functions, are the histone deacetylases (HDAC) and histone acetyltransferases (HAT). These two groups of enzymes modify histones, and this modification is proposed to alter chromatin structure. Acetylated histones are typically localized to active chromatin while deacetylated histones colocalize with transcriptionally inactive chromatin. When these enzymes are directed to a promoter through a DNA binding factor such as YY1, that promoter can be activated or repressed. This review will discuss the recent work dealing with the different proteins that interact with YY1, with particular emphasis on ones that modify chromatin, and how they could be involved in regulating YY1's activities. Publication Types: Review Review, Tutorial PMID: 10452940 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: Genes Cells. 1999 Apr;4(4):197-203. Incorporation of Drosophila TAF110 into the yeast TFIID complex does not permit the Sp1 glutamine-rich activation domain to function in vivo. Keaveney M, Struhl K. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. kevin@hms.harvard.edu BACKGROUND: Acidic activation domains function across eukaryotic species, and hence stimulate transcription by a conserved molecular mechanism. In contrast, glutamine-rich activation domains function in flies, mammals, and fission yeasts but not in the budding yeast Saccharomyces cerevisiae. The glutamine-rich activation domain of Sp1 interacts with TAF110, and it has been suggested that this interaction is important for transcriptional activation. S. cerevisiae does not contain a homologue of TAF110, suggesting a potential mechanism to account for the failure of glutamine-rich activation domains to stimulate transcription. RESULTS: Here, we have artificially recruited Drosophila TAF110 into the yeast TFIID complex by fusing it to yeast TBP. The resulting TFIID complex supports normal cell growth, but it is unable to mediate Sp1-dependent activation. CONCLUSIONS: Thus, the interaction of glutamine-rich activation domains with TAF110 is insufficient for transcriptional activation in vivo, indicating that other targets within the PolII machinery are necessary. PMID: 10336691 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Chem Biol Interact. 1999 Mar 1;118(1):51-67. Differential sensitivity of transcription factors to mustard-damaged DNA. Chen XM, Gray PJ, Cullinane C, Phillips DR. Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia. Nitrogen mustard (bis(2-chloroethyl) methylamine, HN2) inhibited the binding of upstream factors Sp1 and AP2 to their consensus sequences. At concentrations where 50% of the consensus sequence DNA contained at least one lesion, HN2 inhibited formation of the Sp1 complex by 37% (40 microM HN2) and the AP2 complex by 40% (50 microM HN2). The binding of the TATA binding protein (TBP) to the TATA element was also inhibited by HN2, whereas sulphur mustard and the monofunctional sulphur mustard 2-chloroethyl ethyl sulphide (CEES) resulted in a disproportional extent of inhibition with respect to the level of alkylation. The level of alkylation of the TBP oligonucleotide varied significantly at 100 microM drug, with 80, 42 and 15% of HN2, sulphur mustard and CEES, respectively. However, this level of alkylation inhibited formation of the TBP-DNA complex by 70, 70 and 45%, respectively. This differential sensitivity of transcription factors to mustard-induced DNA damage therefore appears to reside dominantly in the stereochemical differences between the specific mustard lesions. PMID: 10227578 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Biochem J. 1999 May 1;339 ( Pt 3):751-8. Transcriptional autorepression of Msx1 gene is mediated by interactions of Msx1 protein with a multi-protein transcriptional complex containing TATA-binding protein, Sp1 and cAMP-response-element-binding protein-binding protein (CBP/p300). Shetty S, Takahashi T, Matsui H, Ayengar R, Raghow R. Department of Biology, The University of Memphis, Memphis, TN 38152, USA. The TATA-less murine Msx1 promoter contains two Msx1-binding motifs, located at -568 to -573 and +25 to +30, and is subject to potent autorepression [Takahashi, Guron, Shetty, Matsui and Raghow (1997) J. Biol. Chem. 272, 22667-22678]. To investigate the molecular mechanism by which Msx1 represses the activity of its own promoter, we transfected C2C12 myoblasts with Msx1-promoter-luciferase constructs and assessed reporter gene activity, with and without the exogenous expression of Msx1. We demonstrate that Msx1-mediated autorepression remained unaffected, regardless of the presence or absence of the Msx1 recognition motifs on the promoter. Furthermore, graded exogenous expression of TATA-binding protein (TBP), Sp1 or cAMP-response-element-binding protein-binding protein (CBP/p300) could counteract the autoinhibitory activity of Msx1. Finally, we demonstrate that Msx1 protein can be immunoprecipitated in a multiprotein complex containing TBP, Sp1 and CBP/p300. We hypothesize that the interaction of Msx1 protein with one or more ubiquitous or tissue-restricted transcription factors mediates transcriptional autorepression of the Msx1 gene. PMID: 10215616 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Oncogene. 1999 Jan 7;18(1):239-47. Cyclin D1 associates with the TBP-associated factor TAF(II)250 to regulate Sp1-mediated transcription. Adnane J, Shao Z, Robbins PD. Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA. We have previously shown that Sp1-mediated transcription is stimulated by Rb and repressed by cyclin D1. The stimulation of Sp1 transcriptional activity by Rb is conferred, in part, through a direct interaction with the TBP-associated factor TAF(II)250. Here we investigated the mechanism(s) through which cyclin D1 represses Sp1. We examined the ability of cyclin D1 to regulate transcription mediated by Gal4-Sp1 fusion proteins, which contain the Gal4 DNA-binding domain and Sp1 trans-activation domain(s). The domain of Sp1 sufficient to confer repression by cyclin D1 was mapped to a region important for interaction with TAF(II)110. We further demonstrate that TAF(II)250-cyclin D1 complexes can be immunoprecipitated from mammalian and baculovirus-infected insect cells and that recombinant GST-TAF(II)250 (amino acids 1-434) associates with cyclin D1 in vitro. Moreover, the overexpression of Rb or CDK4 reduced the level of TAF(II)250-cyclin D1 complex. The amino terminus of cyclin D1 (amino acids 1-100) was sufficient for association with TAF(II)250 and for repressing Sp1-mediated transcription. Taken together, the results suggest that cyclin D1 may regulate transcription by interacting directly or indirectly with TAF(II)250. PMID: 9926939 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: Mol Cell Biol. 1998 Jun;18(6):3234-44. Involvement of TFIID and USA components in transcriptional activation of the human immunodeficiency virus promoter by NF-kappaB and Sp1. Guermah M, Malik S, Roeder RG. Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021, USA. The purified Rel/NF-kappaB (p50/p65) complex and Sp1 markedly activate transcription from the human immunodeficiency virus type 1 (HIV-1) promoter in a highly purified HeLa reconstituted transcription system. Transcriptional activation by NF-kappaB and Sp1 requires both TFIID and the USA fraction. The USA-derived coactivators PC2 and PC4 fully reconstitute the USA coactivator activity, both by repressing the basal level of transcription and by potentiating activator function to yield large increases in the levels of transcription induction. Under limiting concentrations, PC2 and PC4 also show synergistic effects. The C-terminal portion (amino acids 416 to 550) of the p65 subunit of NF-kappaB is a potent activator when assayed as a Gal fusion in the reconstituted transcription system and interacts both with TATA-binding protein (TBP) and with several human TBP-associated factors (TAFs) that include TAFII250. The p65 activation domain mediates transcription activation in the presence of partially reconstituted TFIID species that include a minimal complex containing only TBP and TAFII250. These studies also show that, like USA components, TAFs can serve both to repress TBP-mediated transcription and, following activator interactions, to reverse the repression and effect a net increase in activity. Taken together, these data underscore the importance of both TAFs and specific USA-derived coactivators for optimal activation of the HIV-1 promoter, as well as certain parallels in their overall mechanisms of action. PMID: 9584164 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: EMBO J. 1998 Mar 16;17(6):1768-78. Regional and temporal specialization in the nucleus: a transcriptionally-active nuclear domain rich in PTF, Oct1 and PIKA antigens associates with specific chromosomes early in the cell cycle. Pombo A, Cuello P, Schul W, Yoon JB, Roeder RG, Cook PR, Murphy S. Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. PTF (PSE-binding transcription factor) activates transcription of snRNA and related genes. We investigated its distribution in HeLa nuclei by immunofluorescence, and found it spread throughout the nucleoplasm in small foci. In some cells, PTF is also concentrated in one, or very few, discrete regions (diameter approximately 1.3 micron) that appear during G1 phase and disappear in S phase. Oct1, a transcription factor that interacts with PTF, is also enriched in these domains; RNA polymerase II, TBP and Sp1 are also present. Each domain typically contains 2 or 3 transcription 'factories' where Br-UTP is incorporated into nascent transcripts. Accordingly, we have christened this region the Oct1/PTF/transcription (OPT) domain. It colocalizes with some, but not all, PIKA domains. It is distinct from other nuclear domains, including coiled bodies, gemini bodies, PML bodies and the perinucleolar compartment. A small region on chromosome 6 (band 6p21) containing only approximately 30 Mbp DNA, and chromosomes 6 and 7, associate with the domain significantly more than other chromosomes. The domains may act like nucleoli to bring particular genes on specific chromosomes together to a region where the appropriate transcription and processing factors are concentrated, thereby facilitating the expression of those genes. PMID: 9501098 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: Virology. 1998 Jan 20;240(2):326-37. An Sp1-binding site and TATA element are sufficient to support full transactivation by proximally bound NS1 protein of minute virus of mice. Lorson C, Pearson J, Burger L, Pintel DJ. Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri-Columbia 65212, USA. The minute virus of mice (MVM) P38 Sp1-binding site and TATA box, inserted in an otherwise heterologous plasmid background, could be transactivated to high levels by the MVM NS1 protein targeted proximally to these sequences, demonstrating that these core promoter regulatory elements are sufficient to support essentially wild-type levels of NS1-transactivated expression and suggesting that NS1 may act directly or indirectly with Sp1 and or elements of the general transcription machinery. Accordingly, we show that bacterially generated NS1 can interact strongly, independent of nucleic acid bridging, and most likely directly with Sp1 in vitro and can associate, in a nucleic acid-independent manner, with endogenous Sp1 as it exists in a complex transcriptionally active murine nuclear extract NS1 achieves the same fold activation of an isolated TATA element over its low basal level and can also be demonstrated to interact efficiently and specifically with the general transcription factors TBP and TFIIA (alpha, beta) in vitro. PMID: 9454706 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: Mol Cell Biol. 1997 Dec;17(12):6898-905. Promoter activity of Tat at steps subsequent to TATA-binding protein recruitment. Xiao H, Lis JT, Jeang KT. Laboratory of Molecular Microbiology, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0460, USA. hxiao@atlas.niaid.nih.gov Artificial recruitment of TATA-binding protein (TBP) to many eukaryotic promoters bypasses DNA-bound activator function. The human immunodeficiency virus type 1 (HIV-1) Tat is an unconventional activator that up-regulates transcription from the HIV-1 long terminal repeat (LTR) through binding to a nascent RNA sequence, TAR. Because this LTR and its cognate activator have atypical features compared to a standard RNA polymerase II (RNAP II) transcriptional unit, the precise limiting steps for HIV-1 transcription and how Tat resolves these limitations remain incompletely understood. We thus constructed human TBP fused to the DNA-binding domain of GAL4 to determine whether recruitment of TBP is one rate-limiting step in HIV-1 LTR transcription and whether Tat functions to recruit TBP. As a control, we compared the activity of the adenovirus E1b promoter. Our findings indicate that TBP tethering to the E1b promoter fully effected transcription to the same degree achievable with the potent GAL4-VP16 activator. By contrast, TBP recruitment to the HIV-1 LTR, although necessary for conferring Tat responsiveness, did not bypass a physical need for Tat in achieving activated transcription. These results document that the HIV-1 and the E1b promoters are transcriptionally limited at different steps; the major rate-limiting step for E1b is recruitment of TBP, while activation of the HIV-1 LTR requires steps in addition to TBP recruitment. We suggest that Tat acts to accelerate rate-limiting steps after TBP recruitment. PMID: 9372921 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Virology. 1997 Nov 24;238(2):221-30. Interactions between Tat of HIV-2 and transcription factor Sp1. Pagtakhan AS, Tong-Starksen SE. Department of Medicine, Veterans Administration Medical Center, San Francisco, California, USA. Tat of HIV-2 (Tat-2) requires host cellular factors for optimal function. We show that transactivation by Tat-2 of the HIV promoter requires cis-acting binding sites for Sp1 or Sp1 brought to the promoter via a heterologous system. We demonstrate that an activation domain in Tat-2 consists of one of two potential alpha-helices in the amino-terminal region, the cysteine-rich region, and the core region and that this independent activation domain requires cis-acting Sp1-binding sites for function. Tat-2 interacts with Sp1 in in vitro binding assays, and these interactions require basic residues outside of the Tat-2 activation domain. The regions in Sp1 sufficient for functional synergy with Tat are the Sp1 activation domains, while the DNA-binding region is dispensable. Substitution mutations of a glutamine-rich region in one Sp1 activation domain, which eliminate interactions with a TBP-associated factor, also significantly decrease synergy with Tat. Thus, the functional synergy between Tat-2 and Sp1 localizes to domains in each activator that interact with components of the transcription complex. We suggest that these interactions, rather than direct Tat/Sp1 binding, result in highly processive RNA polymerase II complexes and full-length viral transcripts. PMID: 9400595 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Biol Chem. 1997 Sep;378(9):951-61. Comment in: Biol Chem. 1997 Sep;378(9):935. Helenalin, an anti-inflammatory sesquiterpene lactone from Arnica, selectively inhibits transcription factor NF-kappaB. Lyss G, Schmidt TJ, Merfort I, Pahl HL. Institut fur Pharmazeutische Biologie, Universitat Freiburg, Germany. Alcoholic extracts prepared form Arnicae flos, the collective name for flowerheads from Arnica montana and A. chamissonis ssp. foliosa, are used therapeutically as anti-inflammatory remedies. The active ingredients mediating the pharmacological effect are mainly sesquiterpene lactones, such as helenalin, 11alpha,13-dihydrohelenalin, chamissonolid and their ester derivatives. While these compounds affect various cellular processes, current data do not fully explain how sesquiterpene lactones exert their anti-inflammatory effect. We show here that helenalin, and, to a much lesser degree, 11alpha,13-dihydrohelenalin and chamissonolid, inhibit activation of transcription factor NF-kappaB. This difference in efficacy, which correlates with the compounds' anti-inflammatory potency in vivo, may be explained by differences in structure and conformation. NF-kappaB, which resides in an inactive, cytoplasmic complex in unstimulated cells, is activated by phosphorylation and degradation of its inhibitory subunit, IkappaB. Helenalin inhibits NF-kappaB activation in response to four different stimuli in T-cells, B-cells and epithelial cells and abrogates kappaB-driven gene expression. This inhibition is selective, as the activity of four other transcription factors, Oct-1, TBP, Sp1 and STAT 5 was not affected. We show that inhibition is not due to a direct modification of the active NF-kappaB heterodimer. Rather, helenalin modifies the NF-kappaB/IkappaB complex, preventing the release of IkappaB. These data suggest a molecular mechanism for the anti-inflammatory effect of sesquiterpene lactones, which differs from that of other nonsteroidal anti-inflammatory drugs (NSAIDs), indomethacin and acetyl salicylic acid. PMID: 9348104 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: J Biol Chem. 1997 Oct 17;272(42):26585-94. Transcriptional regulation of the human erythroid 5-aminolevulinate synthase gene. Identification of promoter elements and role of regulatory proteins. Surinya KH, Cox TC, May BK. Department of Biochemistry, University of Adelaide, South Australia, 5005 Australia. We have characterized the 5'-flanking region of the human erythroid-specific 5-amino levulinate synthase (ALAS) gene (the ALAS2 gene) and shown that the first 300 base pairs of promoter sequence gives maximal expression in erythroid cells. Transcription factor binding sites clustered within this promoter sequence include GATA motifs and CACCC boxes, critical regulatory sequences of many erythroid cell-expressed genes. GATA sites at -126/-121 (on the noncoding strand) and -102/-97 were each recognized by GATA-1 protein in vitro using erythroid cell nuclear extracts. Promoter mutagenesis and transient expression assays in erythroid cells established that both GATA-1 binding sites were functional and exogenously expressed GATA-1 increased promoter activity through these sites in transactivation experiments. A noncanonical TATA sequence at the expected TATA box location (-30/-23) bound GATA-1- or TATA-binding protein (TBP) in vitro. Conversion of this sequence to a canonical TATA box reduced expression in erythroid cells, suggesting a specific role for GATA-1 at this site. However, expression was also markedly reduced when the -30/-23 sequence was converted to a consensus GATA-1 sequence (that did not bind TBP in vitro), suggesting that a functional interaction of both factors with this sequence is important. A sequence comprising two overlapping CACCC boxes at -59/-48 (on the noncoding strand) was demonstrated by mutagenesis to be functionally important. This CACCC sequence bound Sp1, erythroid Kruppel-like factor, and basic Kruppel-like factor in vitro, while in transactivation experiments erythroid Kruppel-like factor activated ALAS2 promoter expression through this sequence. A sequence at -49/-39 with a 9/11 match to the consensus for the erythroid specific factor NF-E2 was not functional. Promoter constructs with 5'-flanking sequence from 293 base pairs to 10.3 kilobase pairs expressed efficiently in COS-1 cells as well as in erythroid cells, indicating that an enhancer sequence located elsewhere or native chromatin structure may be required for the tissue-restricted expression of the gene in vivo. PMID: 9334239 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: J Virol. 1997 Oct;71(10):7227-39. TAF-like functions of human cytomegalovirus immediate-early proteins. Lukac DM, Harel NY, Tanese N, Alwine JC. Department of Microbiology and Graduate Group of Molecular Biology, School of Medicine, University of Pennsylvania, Philadelphia 19104-6142, USA. The human cytomegalovirus (HCMV) major immediate-early (IE) proteins IEP86 (IE2(579aa)) and IEP72 (IE1(491aa)) can transcriptionally activate a variety of simple promoters containing a TATA element and one upstream transcription factor binding site. In our previous studies, transcriptional activation was shown to correlate with IEP86 binding to both the TATA-box binding protein (TBP) and the transcription factor bound upstream. IEP72 often synergistically affects the activation by IEP86, although it has not previously been shown to directly interact in vitro with IEP86, TBP, or transcription factors (e.g., Sp1 and Tef-1) bound by IEP86. We report biochemical and genetic evidence suggesting that the major IE proteins may perform a function similar to that of the TBP-associated factors (TAFs) which make up TFIID. Consistent with this model, we found that the major IE proteins interact with a number of TAFs. In vitro, IEP86 bound with drosophila TAF(II)110 (dTAF(II)110) and human TAF(II)130 (hTAF(II)130), while IEP72 bound dTAF(II)40, dTAF(II)110, and hTAF(II)130. Regions on major IE proteins which mediate binding have been defined. In addition, our data indicate that both IEP72 and IEP86 can bind simultaneously to hTAF(II)130, suggesting that this TAF may provide bridging interactions between the two proteins for transcriptional activation and synergy. In agreement, a transcriptional activation mutant of IEP72 is unable to participate in bridging. Confirmation that these in vitro interactions were relevant was provided by data showing that both IEP72 and IEP86 copurify with TFIID and coimmunoprecipitate with purified TFIID derived from infected cell nuclei. To further support a TAF-like function of the IE proteins, we have found that the IE proteins expressed from the intact major IE gene, and to a lesser extent IEP86 alone, can rescue the temperature-sensitive (ts) transcriptional defect in TAF(II)250 in the BHK-21 cell line ts13. Analyses of mutations in the major IE region show that IEP86 is essential for rescue and that IEP72 augments its effect, and that mutations which affect TAF interactions are debilitated in rescue. Our data, showing that the IE proteins can bind with TFIID and rescue a ts transcriptional defect in TAF(II)250, support the model that the IE proteins perform a TAF-like function as components of TFIID. PMID: 9311796 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: J Biol Chem. 1997 Sep 26;272(39):24563-71. Interaction of elongation factors TFIIS and elongin A with a human RNA polymerase II holoenzyme capable of promoter-specific initiation and responsive to transcriptional activators. Pan G, Aso T, Greenblatt J. Banting and Best Department of Medical Research and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1L6, Canada. Affinity chromatography on columns containing the immobilized monomeric transcriptional elongation factor TFIIS or the essential large subunit, Elongin A, of the trimeric elongation factor, Elongin, was used to purify a human RNA polymerase II holoenzyme from HeLa whole cell extract. This holoenzyme contained nearstoichiometric amounts of all the general transcription factors, TFIIB, TFIID (TBP + TAFIIs), TFIIE, TFIIF, and TFIIH, required to accurately initiate transcription in vitro at the adenovirus major late promoter. It behaved as a large complex, slightly smaller than 70 S ribosomes, during gel filtration chromatography, and contained nearly half the TFIID that was present in the extract used for the affinity chromatography. It also contained the cyclin-dependent kinase CDK8, a human homologue of the Saccharomyces cerevisiae holoenzyme subunit SRB10, and many other polypeptides. Efficient interaction of holoenzyme with TFIIS or Elongin A required only the amino-terminal region of either protein. These regions are similar in amino acid sequence but dispensable for TFIIS or Elongin to regulate elongation in vitro by highly purified RNA polymerase II. The transcriptional activators GAL4-VP16 and GAL4-Sp1 activated transcription in vitro by purified holoenzyme in the absence of any additional factors. PMID: 9305922 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: 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] --------------------------------------------------------------- 40: J Biol Chem. 1997 Jul 11;272(28):17654-61. In vitro chromatin assembly of the HIV-1 promoter. ATP-dependent polar repositioning of nucleosomes by Sp1 and NFkappaB. Widlak P, Gaynor RB, Garrard WT. Department of Molecular Biology and Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9140, USA. Nuclease hypersensitive sites exist in vivo in the chromatin of the integrated human immunodeficiency virus (HIV)-1 proviral genome, in the 5'-long terminal repeat (LTR) within the promoter/enhancer region near Sp1 and NFkappaB binding sites. Previous studies from the Kadonaga and Jones laboratories have shown that Sp1 and NFkappaB can establish hypersensitive sites in a truncated form of this LTR when added before in vitro chromatin assembly with Drosophila extracts, thus facilitating subsequent transcriptional activation of a linked reporter gene upon the association of additional factors (Pazin, M. J., Sheridan, P. L., Cannon, K., Cao, Z., Keck, J. G., Kadanaga, J. T., and Jones, K. A. (1996) Genes & Dev. 10, 37-49). Here we assess the role of a full-length LTR and 1 kilobase pair of downstream flanking HIV sequences in chromatin remodeling when these transcription factors are added after chromatin assembly. Using Xenopus laevis oocyte extracts to assemble chromatin in vitro, we have confirmed that Sp1 and NFkappaB can indeed induce sites hypersensitive to DNase I, micrococcal nuclease, or restriction enzymes on either side of factor binding sites in chromatin but not naked DNA. We extend these earlier studies by demonstrating that the process is ATP-dependent when the factors are added after chromatin assembly and that histone H1, AP1, TBP, or Tat had no effect on hypersensitive site formation. Furthermore, we have found that nucleosomes upstream of NFkappaB sites are rotationally positioned prior to factor binding and that their translational frame is registered after binding NFkappaB. On the other hand, binding of Sp1 positions adjacent downstream nucleosome(s). We term this polar repositioning because each factor aligns nucleosomes only on one side of its binding sites. Mutational analysis and oligonucleotide competition each demonstrated that this remodeling required Sp1 and NFkappaB binding sites. PMID: 9211915 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: Nucleic Acids Res. 1997 Jul 1;25(13):2661-71. Identification and characterization of the human XIST gene promoter: implications for models of X chromosome inactivation. Hendrich BD, Plenge RM, Willard HF. Department of Genetics and Center for Human Genetics, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4955, USA. The XIST gene in both humans and mice is expressed exclusively from the inactive X chromosome and is required for X chromosome inactivation to occur early in development. In order to understand transcriptional regulation of the XIST gene, we have identified and characterized the human XIST promoter and two repeated DNA elements that modulate promoter activity. As determined by reporter gene constructs, the XIST minimal promoter is constitutively active at high levels in human male and female cell lines and in transgenic mice. We demonstrate that this promoter activity is dependent in vitro upon binding of the common transcription factors SP1, YY1 and TBP. We further identify two cis -acting repeated DNA sequences that influence reporter gene activity. First, DNA fragments containing a set of highly conserved repeats located within the 5'-end of XIST stimulate reporter activity 3-fold in transiently transfected cell lines. Second, a 450 bp alternating purine-pyrimidine repeat located 25 kb upstream of the XIST promoter partially suppresses promoter activity by approximately 70% in transient transfection assays. These results indicate that the XIST promoter is constitutively active and that critical steps in the X inactivation process must involve silencing of XIST on the active X chromosome by factors that interact with and/or recognize sequences located outside the minimal promoter. PMID: 9185579 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: Mol Cell Biol. 1997 Jun;17(6):2973-84. Accurate positioning of RNA polymerase II on a natural TATA-less promoter is independent of TATA-binding-protein-associated factors and initiator-binding proteins. Weis L, Reinberg D. Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635, USA. Two promoter elements, the TATA element and initiator (Inr), are capable of directing specific transcription initiation of protein-encoding genes by RNA polymerase II (RNAPII). Although binding to the TATA element by the TATA-binding protein (TBP) has been shown to be the initial recognition step in transcription complex formation in vitro, the mechanism through which the basal machinery assembles into a functional complex on TATA-less promoters is controversial. Evidence supporting numerous models of Inr-mediated transcription complex formation exists, including the nucleation of a complex by Inr-binding proteins, a component of the TFIID complex, or a specific upstream activator common to many TATA-less promoters, Sp1. Using various techniques, we have undertaken a systematic analysis of the natural TATA-less human DNA polymerase beta (beta-pol) gene promoter. Although the beta-pol promoter contains upstream Sp1 elements and a functional Inr that binds YY1, neither of these factors is essential for Inr-mediated transcription complex formation. A complex containing TBP, TFIIB, TFIIF, and RNAPII (DBPolF complex) is capable of forming on the promoter in an Inr-dependent manner. A single point mutation within the Inr that affects DBPolF complex formation diminishes beta-pol transcriptional activity. PMID: 9154795 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: Mol Reprod Dev. 1997 Mar;46(3):268-77. Regulation of gene expression in the preimplantation mouse embryo: temporal and spatial patterns of expression of the transcription factor Sp1. Worrad DM, Schultz RM. Department of Biology, University of Pennsylvania, Philadelphia 19104-6018, USA. Activation of the embryonic genome during preimplantation mouse development entails a dramatic reprogramming of the pattern of gene expression. The complement of transcription factors that are present in the early embryo and that must intrinsically be involved in this reprogramming is essentially uncharacterized. We and others have demonstrated that transcription factor Sp1 is present in the mouse oocyte and early cleavage stage preimplantation embryo. Due to Sp1's prominent role in regulating the expression of a vast array of genes that are involved in cell proliferation and differentiation, as well as in general housekeeping functions, we characterized the temporal and spatial patterns of Sp1 expression during preimplantation development. The relative abundance of Sp1 transcripts, as well as transcripts for the TATA box-binding protein TBP, decreases during oocyte maturation and reaches a minimum level in the two-cell stage, after which time the abundance of these transcripts increases progressively to the blastocyst stage. Immunoblotting experiments detect Sp1 species of Mr = 95,000 and 105,000 at all stages of preimplantation development. The amount of Sp1 increases about 8-fold during preimplantation development, and an alpha-amanitin-insensitive increase is observed between G1 and G2 of the one-cell embryo; this increase may reflect the mobilization of a maternal Sp1 transcript. Immunocytochemical experiments also reveal a similar increase in the amount of Sp1 during preimplantation; the nuclear concentration of Sp1 is greater in the trophectoderm cells than in the inner cell mass cells. Finally, gel-shift experiments document an increase during preimplantation development of a DNA-binding activity that is likely due to Sp1. These increases in the abundance of the Sp1 protein and an Sp1-like DNA-binding activity parallel increases in the rate of transcription that occur during preimplantation development. PMID: 9041129 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: Science. 1997 Feb 7;275(5301):829-31. Selective use of TBP and TFIIB revealed by a TATA-TBP-TFIIB array with altered specificity. Tansey WP, Herr W. Cold Spring Harbor Laboratory, 1 Bungtown Road, Post Office Box 100, Cold Spring Harbor, NY 11724, USA. Interaction between the TATA box-binding protein TBP and TFIIB is critical for transcription in vitro. An altered-specificity TBP-TFIIB interaction was rationally designed and linked in sequence to an altered-specificity TATA box-TBP interaction to study how TBP and TFIIB function together to support transcription in human cells. The activity of this altered-specificity TATA-TBP-TFIIB array demonstrated that many activators use the known TBP-TFIIB interaction to stimulate transcription. One activator, however, derived from a glutamine-rich activation domain of Sp1, activated transcription independently of this interaction. These results reveal that selectivity in activator function in vivo can be achieved through differential use of TBP and TFIIB. PMID: 9012349 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: Bioorg Med Chem. 1997 Jan;5(1):205-15. Synthetic inhibitors of regulatory proteins involved in the signaling pathway of the replication of human immunodeficiency virus 1. Otsuka M, Fujita M, Sugiura Y, Yamamoto T, Inoue J, Maekawa T, Ishii S. Institute for Chemical Research, Kyoto University, Japan. NF-kappa B, HIV-EP1, Sp1, and E1A are transcriptional proteins involved in the long terminal repeat-directed expression of HIV-1. The inhibitory effect of 18 dimethylaminopyridine-based compounds against these regulatory proteins was studied. Experiments using NF-kappa B-beads showed that histidine-pyridine-histidine compounds and their zinc complexes are inhibitory not only for the NF-kappa B-DNA binding, but also for the binding of NF-kappa B with the inhibitory protein I kappa B. Discriminative inhibition of the DNA binding of two distinct C2H2 type zinc finger proteins HIV-EP1 and Sp1 was also attempted using the synthetic compounds. Whereas some compounds inhibited the DNA binding of both HIV-EP1 and Sp1 at 300 microM, others preferentially and completely inhibited HIV-EP1 without much suppression of Sp1. Mercapto compounds were more potent and uniformly inhibitory against both HIV-EP1 and Sp1 at 30 microM. Disulfide compounds were also remarkably inhibitory against HIV-EP1 and Sp1 also at 30 microM whereas the shorter-chain disulfides 7 and 9 were effective only for HIV-EP1. S-Alkyl derivatives preferentially inhibited HIV-EP1 at 300 microM. The dimethylamino compound was the sole compound inhibitory only against Sp1, being non-inhibitory against HIV-EP1. Relevant combinations of these inhibitors would allow us to inhibit NF-kappa B, HIV-EP1, and Sp1 in any combinations. Inhibition of the TBP binding of C4 type zinc finger protein adenovirus E1A was also examined. It was found that two compounds induced, at 50 mM concentration, effective inhibition of the TBP binding of E1A, demonstrating that it is possible in principle to inhibit the protein-protein interaction of zinc finger proteins. PMID: 9043672 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13611-6. Molecular cloning and analysis of two subunits of the human TFIID complex: hTAFII130 and hTAFII100. Tanese N, Saluja D, Vassallo MF, Chen JL, Admon A. Department of Microbiology and Kaplan Cancer Center, New York University Medical Center, NY 10016, USA. Transcription factor TFIID is a multiprotein complex composed of the TATA box-binding protein (TBP) and multiple TBP-associated factors (TAFs). TFIID plays an essential role in mediating transcriptional activation by gene-specific activators. Numerous transcriptional activators have been characterized from mammalian cells; however, molecular analysis of the components of mammalian TFIID has been incomplete. Here we describe isolation of cDNAs encoding two TAF subunits of the human transcription factor TFIID. The first cDNA is predicted to encode the C-terminal 947 residues of the 130-kDa human TAF subunit, hTAFII130. The second cDNA encodes the C-terminal 801 residues of the 100-kDa subunit, hTAFII100. Recombinant TAFs expressed in human cells by transient transfections are capable of associating with the endogenous TAFs and TBP to form a TFIID complex in vivo. Protein binding experiments demonstrate that hTAFII130, like its Drosophila homolog dTAFII110, interacts with the glutamine-rich activation domains of the human transcription factor Sp1. Furthermore, hTAFII130 shows reduced binding to the Sp1 mutants with impaired ability to activate transcription, suggesting a role for hTAFII130 as a direct coactivator target for Sp1. PMID: 8942982 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: Nucleic Acids Res. 1996 Nov 1;24(21):4281-8. Transcriptional repression by p53 involves molecular interactions distinct from those with the TATA box binding protein. Farmer G, Friedlander P, Colgan J, Manley JL, Prives C. Department of Biological Sciences, Columbia University, New York, NY 10027, USA. In addition to serving a role as a DNA binding-dependent transcriptional activator, p53 has been reported to repress a variety of promoters that lack p53 binding sites. Data from recent studies have suggested that this activity is mediated via an interaction between p53 and the TATA box binding protein (TBP). To investigate the functional relevance of this interaction in vivo, we have performed transient transfection assays in Drosophila Schneider cells. Wild-type p53 was found to repress expression from TATA box- but not initiator (Inr)-containing promoters activated by GAL4-VP16, GAL4-ftzQ or Sp1. A mutant p53(His175), defective in DNA binding and transcriptional activation, also inhibited TATA-dependent transcription activated by Sp1. However, p53 was unable to repress a basal TATA promoter stimulated by overexpression of TBP. Furthermore, overexpression of TBP failed to rescue the p53-mediated repression of activated transcription and a p53 mutant with its N-terminal TBP interaction domain intact, but defective in transcriptional activation and binding to TBP-associated factors (TAFs), was similarly defective in transcriptional repression. These data suggest that a p53-TBP interaction is not sufficient for transcriptional repression by p53 and that repression involves an interaction between p53 and other factors, such as TAFs, that are required for activated but not basal transcription. We suggest that p53-mediated repression results from squelching of a factor limiting for activated transcription from TATA- but not Inr-containing promoters. PMID: 8932384 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: EMBO J. 1996 Oct 15;15(20):5659-67. An inhibitor domain in Sp3 regulates its glutamine-rich activation domains. Dennig J, Beato M, Suske G. Institut fur Molekularbiologie und Tumorforschung, Germany. Sp3 is a ubiquitously expressed human transcription factor closely related to Sp1 and Sp4. All three proteins contain a highly conserved DNA binding domain and two glutamine-rich regions, suggesting that they possess similar activation functions. In our previous experiments, however, Sp3 failed to activate transcription. Instead, it repressed Sp1-mediated transcriptional activation, suggesting that it is an inhibitory member of this family of regulatory factors. Here we show that Sp3 can also act as a positive regulator of transcription. The glutamine-rich domains on their own have a strong activation function and interact with the TATA box binding protein (TBP)-associated factor dTAFII110. However, in full-length Sp3 as well as in Gal4-Sp3 fusion proteins, both activation domains are silenced by an inhibitory domain located between the second glutamine-rich region and the DNA binding domain. The inhibitory domain completely suppressed transcriptional activation when fused to a heterologous glutamine-rich domain but only moderately suppressed transcription when linked to an acidic activation domain. Site-directed mutagenesis identified a stretch of highly charged amino acid residues essential for inhibitor function. Substitution of the amino acid triplet KEE by alanine residues within this region changed the almost transcriptionally inactive Sp3 into a strong activator. Our results suggest that the transcriptional activity of Sp3 might be regulated in vivo by relief of inhibition. PMID: 8896459 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: Mol Cell Biol. 1996 Aug;16(8):4295-304. Functional interaction between p53, the TATA-binding protein (TBP), andTBP-associated factors in vivo. Farmer G, Colgan J, Nakatani Y, Manley JL, Prives C. Department of Biological Sciences, Columbia University, New York, New York 10027, USA. The transcriptional activator p53 is known to interact with components of the general transcription factor TFIID in vitro. To examine the relevance of these associations to transcriptional activation in vivo, plasmids expressing a p53-GAL4 chimera and Drosophila TATA-binding protein (dTBP) were transfected into Drosophila Schneider cells. p53-GAL4 and dTBP displayed a markedly synergistic effect on activated transcription from a GAL4 site-containing reporter that was at least 10-fold greater than observed with other activators tested. A mutant p53 previously shown to be defective in both transcriptional activation in vivo and in binding to TBP-associated factors (TAFs) in vitro, although still capable of binding dTBP, did not cooperate with dTBP, suggesting that TAFs may contribute to this synergy. Providing further support for this possibility, transfected dTBP assembled into rapidly sedimenting complexes and could be immunoprecipitated with anti-TAF antibodies. While overexpression of any of several TAFs did not affect basal transcription, in either the presence or the absence of cotransfected dTBP, overexpression of TAFII230 inhibited transcriptional activation mediated by p53-GAL4 as well as by GAL4-VP16 and Sp1. Overexpression of TAFII40 and TAFII60 also inhibited activation by p53-GAL4 but had negligible effects on activation by GAL4-VP16 and Sp1, while TAFII110 did not affect any of the activators. TAF-mediated inhibition of activated transcription could be rescued by high levels of exogenous dTBP, which also restored full synergy. These data demonstrate for the first time that functional interactions can occur in vivo between TBP, TAFs, and p53. PMID: 8754830 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 50: 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] --------------------------------------------------------------- 51: J Virol. 1996 May;70(5):2922-9. Inhibition of basal transcription by poliovirus: a virus- encoded protease (3Cpro) inhibits formation of TBP-TATA box complex in vitro. Yalamanchili P, Harris K, Wimmer E, Dasgupta A. Department of Microbiology and Immunology, University of California, Los Angeles, School of Medicine, 90024-1747, USA. Host cell RNA polymerase II (pol II)-mediated transcription is inhibited by poliovirus infection. We demonstrate here that both TATA- and initiator-mediated basal transcription is inhibited in extracts prepared from poliovirus-infected HeLa cells. This inhibition can be reproduced by incubation of uninfected HeLa cell extracts with purified, recombinant poliovirus protease, 3Cpro. Transient-transfection assays demonstrate that 3Cpro, in the absence of other viral proteins, is able to inhibit cellular pol II-mediated transcription in vivo. Three lines of evidence suggest that inactivation of TATA-binding protein (TBP) is the major cause of inhibition of basal transcription by poliovirus. First, RNA pol II transcription in poliovirus-infected cell extract is fully restored by bacterially expressed TBP. Second, addition of purified TBP restores transcription in heat-treated nuclear extracts from mock- and virus-infected cells to identical levels. Finally, using a gel mobility shift assay, we demonstrate that incubation of TBP with the viral protease (3Cpro) inhibits its ability to bind TATA sequence in vitro. These results suggest that inhibition of pol II transcription in mammalian cells infected with poliovirus is, at least in part, due to the inability of modified TBP to bind pol II promoter sequences. PMID: 8627767 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 52: J Mol Recognit. 1996 Mar-Apr;9(2):75-87. Specific targeting of protein-DNA complexes by DNA-reactive drugs (+)-CC-1065 and pluramycins. Henderson D, Hurley LH. Drug Dynamics Institute, College of Pharmacy, University of Texas, Austin 78712, USA. To gain insight into the interactions between transcriptional factor proteins and DNA, the DNA-reactive drugs (+)-CC-1065 and pluramycin were used to target specific protein-DNA complexes. The structural features of the complex between the transcriptional activator Sp1 and the 21-base-pair repeat of the early promoter region of SV40 DNA were examined using hydroxyl-radical footprinting; (+)-CC-1065, a sequence-specific minor groove bending probe; and circularization experiments. The results show that the 21-base-pair repeat region has an intrinsically in-phase bent structure that is stabilized upon saturation Sp1 binding by protein-DNA and protein-protein interactions to produce a looping structure. The intercalating drug pluramycin was used to probe the structural details of the interaction between the TATA binding protein (TBP) and the TATA box DNA sequence. TBP, which directs initiation of RNA transcription, exhibits two-fold symmetry and apparently interacts with the TATA box in a symmetrical fashion. However, the interaction results in an asymmetric effect, in that transcription is initiated only in the downstream direction. Using pluramycin as a probe, it was determined that TBP binding to the human myoglobin TATA sequences enhances pluramycin reactivity at a site immediately downstream of the TATA box. The implications on transcriptional control of ternary complexes comprised of transcriptional factors, DNA, and DNA-reactive compounds will be presented. Publication Types: Review Review, Tutorial PMID: 8877797 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 53: J Virol. 1996 Feb;70(2):1191-202. The major transcriptional transactivation domain of simian virus 40 large T antigen associates nonconcurrently with multiple components of the transcriptional preinitiation complex. Johnston SD, Yu XM, Mertz JE. McArdle Laboratory for Cancer Research, University of Wisconsin, Medical School, Madison 53706-1599, USA. Simian virus 40 (SV40) large T antigen (Tag) is a promiscuous transcriptional transactivator; however, its mechanism of transactivation remains unknown. Recent studies have suggested the possible involvement of protein-protein interactions with TBP, the TATA box-binding protein of TFIID, and TEF-1, an enhancer-binding factor. We show here that (i) the Tag domain containing amino acids 133 to 249 directly interacts with the general transcription factor TFIIB, the activator protein Sp1, and the 140-kDa subunit of RNA polymerase II, as well as with TBP and TEF-1; (ii) these interactions can also occur when these transcription factors are present in their functional states in cellular extracts; (iii) binding of Tag to TBP is eliminated by preincubation of TBP either at 48 degrees C or with the adenovirus 13S E1a protein; (iv) this domain of Tag cannot bind concurrently to more than one of these transcription factors; and (v) the substitution of Tag amino acid residues 173 and 174 inactivates the ability of this Tag domain both to associate with any of these transcription factors and to transactivate the SV40 late promoter. Thus, we conclude that SV40 Tag probably does not transactivate via the concurrent interaction with multiple components of the preinitiation complex. Rather, we hypothesize that transactivation by Tag may primarily occur by removing or preventing the binding of factors that inhibit the formation of preinitiation complexes. PMID: 8551580 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 54: Gene Expr. 1996;5(4-5):217-28. Sequential steps in Tat trans-activation of HIV-1 mediated through cellular DNA, RNA, and protein binding factors. Gatignol A, Duarte M, Daviet L, Chang YN, Jeang KT. Unite 332 INSERM, Institut Cochin de Genetique Moleculaire, Paris, France. The regulation of HIV expression is controlled by the activity of the Long Terminal Repeat (LTR). Trans-activation by the virally encoded Tat protein is one of the main mechanisms of LTR activation. Tat binds to its target, TAR RNA, and cellular proteins that bind the LTR, Tat, or TAR RNA are important components of the trans-activation process. We will review the factors that have been characterized for a possible involvement in this mechanism. Whereas LTR binding proteins consist of Sp1 and TBP, a large number of factors that bind TAR RNA have been isolated. We have previously cloned two of them by RNA probe recognition: TRBP and La. We have shown that the in vitro and in vivo binding of TRBP to TAR RNA correlates with a constant expression of the protein during HIV-1 infection. Several proteins that interact with Tat have mainly positive, but some negative, effects on trans-activation. Genetic studies have defined that human chromosome 12 encodes a protein that will allow trans-activation in rodent cells. The binding and the functional data about these proteins suggest sequential steps for the Tat trans-activation mechanism. Each of these intracellular molecular events could be the target for molecular intervention against the virus. PMID: 8723388 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 55: Mol Cell Biol. 1995 Nov;15(11):6283-90. Subregions of the adenovirus E1A transactivation domain target multiple components of the TFIID complex. Geisberg JV, Chen JL, Ricciardi RP. Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104, USA. Transcriptional activation by the adenovirus E1A 289R protein requires direct contacts with the TATA box-binding protein (TBP) and also displays a critical requirement for TBP-associated factors (TAFs) (T.G. Boyer and A. J. Berk, Genes Dev. 7:1810-1823, 1993; J. V. Geisberg, W. S. Lee, A. J. Berk, and R. P. Ricciardi, Proc. Natl. Acad. Sci. USA 91:2488-2492, 1994; W. S. Lee, C. C. Kao, G. O. Bryant, X. Liu, and A. J. Berk, Cell 67:365-376, 1991; and Q. Zhou, P. M. Lieberman, T. G. Boyer, and A. J. Berk, Genes Dev. 6:1964-1974, 1992). In this report, we demonstrate that the activation domain of E1A (CR3) specifically binds to two TAFs, human TAFII250 (hTAFII250) and Drosophila TAFII110 (dTAFII110). These interactions can take place both in vivo and in vitro and require the carboxy-terminal region of CR3; the zinc finger region of CR3, which binds TBP, is not needed to bind these TAFs. We mapped the E1A-binding sites on hTAFII250 to an internal region that contains a number of structural motifs, including an HMG box, a bromodomain, and direct repeats. This represents the first demonstration that hTAFII250 may serve as a target of a transcriptional activator. We also mapped the E1A binding on dTAFII110 to its C-terminal region. This is of significance since, by contrast, Sp1-mediated activation requires binding to the N-terminal domain of dTAFII110. Thus, distinct surfaces of dTAFII110 can serve as target sites for different activators. Our results indicate that E1A may activate transcription, in part, through direct contacts of the CR3 subdomains with selected components of the TFIID complex. PMID: 7565781 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 56: Oncogene. 1995 Oct 5;11(7):1299-307. p53 represses SV40 transcription by preventing formation of transcription complexes. Perrem K, Rayner J, Voss T, Sturzbecher H, Jackson P, Braithwaite A. Division of Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia. There is now much evidence to suggest that the p53 tumour suppressor protein functions to monitor the integrity of the genome. When DNA damage is detected, p53 suppresses cell growth to allow repair or directs the cell into apoptosis. The mechanism of action of p53 is as yet unclear but recent evidence has accumulated to suggest that p53 might act by regulating gene expression. Consistent with this model, p53 can both activate and repress a number of viral and cellular promoters. p53 has also been shown to bind to the CCAAT-binding Factor and TATA-binding protein (TBP), and there is direct evidence that p53 represses in vitro transcription by preventing TBP from binding DNA. We now provide evidence that p53 can repress transcription from the SV40 promoter by disrupting DNA/protein complexes involving transcription factor Sp1. PMID: 7478550 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 57: Nucleic Acids Res. 1995 Sep 11;23(17):3473-80. TBP binding and the rate of transcription initiation from the human beta-globin gene. Antoniou M, de Boer E, Spanopoulou E, Imam A, Grosveld F. Laboratory of Gene Structure and Expression, National Institute for Medical Research, London, UK. DNA-protein interaction studies in vitro revealed several factors binding over the TATA box and the region of transcription initiation (cap) site of the human beta-globin promoter; TATA binding protein TBP at -30, Sp1 at -19, GATA-1 at -12 and +5, YY1 at -9 and a novel factor C1 over the site of initiation (-4 to +7). Point mutants which specifically abolish the binding of each of these proteins were tested in a beta-globin locus control region (LCR) construct which allows quantitative comparisons at physiological levels of transcription. Only mutants which drastically affect the binding of TBP resulted in decreased levels of transcription. A threshold value of TBP binding of 15-30% of wild type was sufficient to give normal levels of transcription. This indicates that the association of TF IID with the TATA box is not limiting in the rate of initiation of transcription. PMID: 7567458 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 58: Mol Reprod Dev. 1995 Jun;41(2):232-8. Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Ho Y, Wigglesworth K, Eppig JJ, Schultz RM. Department of Biology, University of Pennsylvania, Philadelphia 19104-6018, USA. Simplex optimization has generated several media that have improved the development of mouse preimplantation embryos in vitro. One objective of this study was to compare the development of preimplantation mouse embryos in one of these computer-optimized media, KSOM, with embryos that developed in vivo, in terms of the relative abundances of specific mRNAs involved in metabolism, transcription, and cell proliferation. First, however, since studies have indicated an improvement of other simple embryo culture media by addition of amino acids, the effects of the addition of amino acids to KSOM (KSOM/AA) on preimplantation development were assessed. We find that addition of both essential and nonessential amino acids to KSOM augments development in vitro, as compared to development supported by KSOM without amino acids. This augmentation is observed starting at the blastocyst stage, and is associated with increased rate of development to the blastocyst stage, increased frequency of hatching, and increased number of cells in the blastocysts. Reverse-transcription PCR was then used to assess the relative abundance of mRNAs for actin, glyceraldehyde-3-phosphate dehydrogenase, Na+, K(+)-ATPase, Sp1, TATA box-binding protein TBP, IGF-I, IGF-II, IGF-I receptor, and IGF-II receptor in embryos that developed in vivo and in vitro using KSOM/AA. Eight out of 9 of these mRNAs were present in the 8-cell embryos and blastocysts raised in KSOM/AA in amounts that were indistinguishable from those in embryos that developed in vivo. It is concluded that KSOM/AA provides an environment in which preimplantation mouse embryos can undergo development that is quantitatively similar to that occurring in vivo. PMID: 7654376 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 59: Virology. 1995 Apr 20;208(2):753-61. Induction of Sp1 phosphorylation and NF-kappa B-independent HIV promoter domain activity in T lymphocytes stimulated by okadaic acid. Vlach J, Garcia A, Jacque JM, Rodriguez MS, Michelson S, Virelizier JL. Unite d'Immunologie Virale, Institut Pasteur, Paris, France. In contrast to the purely enhancer-dependent effect of cytokines such as TNF on the activity of the HIV regulatory region (LTR), we observed that okadaic acid (OKA) activates HIV transcription through both the enhancer, responding to the factor NF-kappa B, and the promoter domain of the LTR. The inducibility of HIV LTR-driven luciferase expression constructs in lymphoblastoid cells stimulated by OKA depended on both functional Sp1 binding elements and the ability of the TATA box to bind the protein TBP. In both transformed and normal lymphocytes, OKA stimulation induced intense phosphorylation of the constitutively expressed Sp1 protein in the nucleus, a property of OKA not shared by TNF, phorbol ester, or PHA and interleukin 2. Responsiveness of LTR constructs deleted of kappa B elements to HIV Tat expression was increased upon OKA but not TNF stimulation. Our results suggest that SP1 phosphorylation induced by OKA, a selective inhibitor of the serine-threonine phosphatase PP2A, facilitates the formation of a transcription complex involving general transcription factors, HIV Tat, and Sp1 proteins. The formation of this complex would increase, independently of an in synergy with NF-kappa B, the low basal activity of the HIV LTR observed in normal T lymphocytes. PMID: 7747447 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 60: Mol Cell Biol. 1995 Mar;15(3):1554-63. Transcriptional activators differ in their responses to overexpression of TATA-box-binding protein. Sadovsky Y, Webb P, Lopez G, Baxter JD, Fitzpatrick PM, Gizang-Ginsberg E, Cavailles V, Parker MG, Kushner PJ. Metabolic Research Unit, University of California, San Francisco 94143. We investigated how overexpression of human TATA-box-binding protein (TBP) affects the action of estrogen receptor (ER) and compared the response with that of other activators. When ER activates a simple promoter, consisting of a response element and either the collagenase or tk TATA box, TBP overexpression potentiates transcription. TBP potentiates only estrogen-induced and not basal transcription and does so independent of spacing between response element and TATA box. TBP overexpression also reduces autoinhibition by overexpressed ER, suggesting that one target of the autoinhibition may be TBP itself. Both AF-1 and AF-2 domains of ER are potentiated by TBP, and each domain binds TBP in vitro. Like ER, chimeric GAL4/VP16 and GAL4/Tat activators are also potentiated by TBP, as is the synergistic activation by ER and GAL4/VP16 on a complex promoter. Unlike ER, GAL4/Sp1 and GAL4/NF-I become less potent when TBP is overexpressed. Furthermore, synergy between ER and Sp1 or between ER and NF-I, whether these are supplied by transfected GAL4 fusions or by the endogenous genes, is inhibited by TBP overexpression. Thus, ER resembles VP16 in response to TBP overexpression and is different from Sp1 and NF-I, which predominate over ER in setting the response on complex promoters. PMID: 7862148 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 61: Mol Cell Biol. 1995 Feb;15(2):983-8. The glutamine-rich activation domains of human Sp1 do not stimulate transcription in Saccharomyces cerevisiae. Ponticelli AS, Pardee TS, Struhl K. Department of Biochemistry, State University of New York at Buffalo, 14214. Eukaryotic transcriptional activators have been classified on the basis of the characteristics of their activation domains. Acidic activation domains, such as those in the yeast GAL4 or GNC4 proteins and the herpes simplex virus activator VP16, stimulate RNA polymerase II transcription when introduced into a variety of eukaryotic cells. This species interchangeability demonstrates that the mechanism by which acidic activation domains function is highly conserved in the eukaryotic kingdom. To determine whether such a conservation of function exists for a different class of activation domain, we have tested whether the glutamine-rich activation domains of the human transcriptional activator Sp1 function in the yeast Saccharomyces cerevisiae. We report here that the glutamine-rich domains of Sp1 do not stimulate transcription in S. cerevisiae, even when accompanied by human TATA-box binding protein (TBP) or human-yeast TATA-box binding protein hybrids. Thus, in contrast to the case for acidic activation domains, the mechanism by which glutamine-rich domains stimulate transcription is not conserved between S. cerevisiae and humans. PMID: 7823962 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 62: Science. 1995 Jan 27;267(5197):531-6. Cloning of an intrinsic human TFIID subunit that interacts with multiple transcriptional activators. Chiang CM, Roeder RG. Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, NY 10021. TFIID is a multisubunit protein complex comprised of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). The TAFs in TFIID are essential for activator-dependent transcription. The cloning of a complementary DNA encoding a human TFIID TAF, TAFII55, that has no known homolog in Drosophila TFIID is now described. TAFII55 is shown to interact with the largest subunit (TAFII230) of human TFIID through its central region and with multiple activators--including Sp1, YY1, USF, CTF, adenoviral E1A, and human immunodeficiency virus-type 1 Tat proteins--through a distinct amino-terminal domain. The TAFII55-interacting region of Sp1 was localized to its DNA-binding domain, which is distinct from the glutamine-rich activation domains previously shown to interact with Drosophila TAFII110. Thus, this human TFIID TAF may be a co-activator that mediates a response to multiple activators through a distinct mechanism. PMID: 7824954 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 63: EMBO J. 1995 Jan 16;14(2):329-40. The bovine papillomavirus 1 E2 protein contains two activation domains: one that interacts with TBP and another that functions after TBP binding. Steger G, Ham J, Lefebvre O, Yaniv M. Unite des Virus Oncogenes, URA 1644 du CNRS, Institut Pasteur, Paris, France. The E2 transactivator of bovine papillomavirus type-1 is unable to activate minimal promoters in vivo that contain only E2 binding sites and a TATA box. This block can be overcome by over-expression of human TATA binding protein (TBP) or by the addition of either SP1 binding sites or an initiator element to the promoter, suggesting that the binding of TFIID may normally be a rate-limiting step for activation by E2. Surprisingly, purified E2 and TBP bind co-operatively to DNA in vitro when the sites are closely spaced. E2 does not affect the on rate of association but reduces the off rate. The E2 region responsible for this effect is located in the hinge region that links the classic transactivation and DNA binding domains. We demonstrate that the TBP stabilizing domain contributes in vivo to co-operativity with co-expressed TBP and to activation of the major late minimal promoter (MLP) containing E2 sites. In contrast, promoters with SP1 sites are activated to wild-type levels by such a mutant. This promoter specificity is also evident in vitro. A truncated E2 mutant, lacking the classic transactivation domain but containing the TBP stabilizing domain, stimulates transcription of the MLP in vitro, but does not activate promoters with SP1 sites. In conclusion, our results show that the E2 transactivation domain has a modular structure. We have identified one domain which probably acts at an early step in the assembly of the pre-initiation complex and which is involved in reducing the dissociation rate of bound TBP in vitro. The classic N-terminal activation domain of E2 might affect one or several step(s) in the assembly of the preinitiation complex occurring after the binding of TFIID. PMID: 7835344 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 64: EMBO J. 1995 Jan 16;14(2):321-8. Novel mechanism and factor for regulation by HIV-1 Tat. Zhou Q, Sharp PA. Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139. Tat regulation of human immunodeficiency virus (HIV) transcription is unique because of its specificity for an RNA target, TAR, and its ability to increase the efficiency of elongation by polymerase. A reconstituted reaction that is Tat-specific and TAR-dependent for activation of HIV transcription has been used to identify and partially purify a cellular activity that is required for trans-activation by Tat, but not by other activators. In the reaction, Tat stimulates the efficiency of elongation by polymerase, whereas Sp1 and other DNA sequence-specific transcription factors activate the rate of initiation. Furthermore, while TATA binding protein (TBP)-associated factors (TAFs) in the TFIID complex are required for activation by transcription factors, they are dispensable for Tat function. Thus, Tat acts through a novel mechanism, which is mediated by a specific host cellular factor, to stimulate HIV-1 gene expression. PMID: 7835343 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 65: Cell. 1994 Oct 7;79(1):93-105. Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators. Chen JL, Attardi LD, Verrijzer CP, Yokomori K, Tjian R. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720-3202. We previously reported that transcriptional regulators can bind selected TAF subunits of the TFIID complex. However, the specificity and function of individual TAFs in mediating transcriptional activation remained unknown. Here we report the in vitro assembly and transcriptional properties of TBP-TAF complexes reconstituted from the nine recombinant subunits of Drosophila TFIID. A minimal complex containing TBP and TAFII250 directs basal but not activator-responsive transcription. By contrast, reconstituted holo-TFIID supports activation by an assortment of activators. The activator NTF-1, which binds TAFII150, stimulates transcription with a complex containing only TBP, TAFII250, and TAFII150, whereas Sp1 binds and additionally requires TAFII110 for activation. Interestingly, TAFII150 enhances Sp1 activation even though this subunit does not bind directly to Sp1. These results establish that specific subcomplexes of TFIID can mediate activation by different classes of activators and suggest that TAFs perform multiple functions during activation. PMID: 7923382 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 66: Genes Dev. 1994 Oct 1;8(19):2313-23. Drosophila TFIIA directs cooperative DNA binding with TBP and mediates transcriptional activation. Yokomori K, Zeidler MP, Chen JL, Verrijzer CP, Mlodzik M, Tjian R. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720-3202. Drosophila transcription factor IIA (TFIIA) is composed of three subunits (30, 20, and 14 kD) that function during initiation of transcription. We reported previously the characterization of cDNAs that encode a precursor (dTFIIA-L) of the Drosophila TFIIA 30- and 20-kD subunits. In the absence of the smallest subunit, dTFIIA-S (14 kD), the unprocessed large subunit failed to exhibit any detectable promoter binding or transcriptional activity. Here, we report the molecular cloning and expression of dTFIIA-S, which has allowed the assembly of holo-dTFIIA (dTFIIA-L/S). Subunit interaction studies indicate that dTFIIA-S binds to an amino-terminal domain of dTFIIA-L, which likely corresponds to the endogenous 30-kD processed species. In addition, both dTFIIA-S and the carboxy-terminal domain of dTFIIA-L, which corresponds to the 20-kD species, independently interact weakly with the TATA-binding protein (TBP). In contrast, the holo-dTFIIA (L/S) binds TBP with high affinity. The dTFIIA-L/S complex also binds cooperatively with TBP to TATA box DNA sequences, generating an extended DNase footprint pattern. The reconstituted holo-dTFIIA is able to stimulate basal transcription of several core promoter templates. Interestingly, dTFIIA-L/S is also able to significantly enhance transcriptional activation by upstream transcription factors including Sp1, VP16, and NTF-1. These results suggest that dTFIIA is a multifunctional transcription factor capable of influencing DNA binding as well as interactions with the basal machinery, thereby enhancing activator-dependent transcription. PMID: 7958898 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 67: J Virol. 1994 Aug;68(8):5184-93. Transcriptional activation by the human cytomegalovirus immediate-early proteins: requirements for simple promoter structures and interactions with multiple components of the transcription complex. Lukac DM, Manuppello JR, Alwine JC. Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia 19104. We have utilized a number of well-defined, simple, synthetic promoters (upstream factor binding sites and TATA elements) to analyze the activation mechanisms of the human cytomegalovirus immediate-early (IE) proteins. We found that the 86-kDa IE protein (known as IEP86, IE2(559aa), or ppUL122a) can recognize and activate a variety of simple promoters, in agreement with the observation that it is a promiscuous activator. However, in the comparison of otherwise identical promoters IEP86 does have preferences for specific TATA elements (hsp70 > adenovirus E2 > simian virus 40 early) and specific upstream transcription factor binding sites (CAAT > SP1 approximately Tef-1 > ATF; no activation with AP1 or OCT). In contrast, the 72-kDa IE protein (known as IEP72, IE1(491aa), or ppUL123) alone did not significantly activate the simple promoters under our experimental conditions. However, each promoter activated by IEP86 was synergistically affected by the addition of IEP72. In addition, the 55-kDa IE protein (IEP55, a splice variant form of IE2, IE2(425aa), or ppUL122b) repeatedly had a negative effect, downregulating the activation of promoters caused by IEP86 and the synergy of IEP86 and IEP72. We show that the ability of IEP86 to activate many simple promoters correlates not only with its previously described ability to interact with the TATA-binding protein (TBP) (B. A. Furnari, E. Poma, T. F. Kowalik, S.-M. Huong, and E.-S. Huang, J. Virol. 67:4981-4991, 1993; C. Hagemeier, S. Walker, R. Caswell, T. Kouzarides, and J. Sinclair, J. Virol. 66:4452-4456, 1992; R. Jupp, S. Hoffman, R. M. Stenberg, J. A. Nelson, and P. Ghazal, J. Virol. 67:7539-7546, 1993) but also with its ability to interact with the transcription factors which bind to the upstream element of promoters it activated (e.g., SP1 and Tef-1 but not Oct-1). This ability to have multiple interactions with the promoter complex may be crucial for transcriptional activation, since the IE proteins cannot activate promoters having only a TATA element or only an upstream transcription factor binding site. In addition, we show that proteins which bind IEP86 also bind to IEP55. Thus, the negative effect on transcription noted with IEP55 may be the result of competition with IEP86 for interaction with the promoter complex. The synergy caused by IEP72 appears to be mediated by a more indirect mechanism. This is suggested by our observation that IEP72 could not bind to any of the proteins tested (TBP, Tef-1, or Oct-1) or to IEP86. PMID: 8035517 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 68: Development. 1994 Aug;120(8):2347-57. Regulation of gene expression in the mouse oocyte and early preimplantation embryo: developmental changes in Sp1 and TATA box-binding protein, TBP. Worrad DM, Ram PT, Schultz RM. Department of Biology, University of Pennsylvania, Philadelphia 19104-6018. We previously demonstrated that an Sp1-dependent reporter gene is preferentially expressed in G2 of the 1-cell mouse embryo following microinjection of the male pronucleus when compared to microinjection of the female pronucleus (P.T. Ram and R.M. Schultz, 1993, Dev. Biol. 156, 552-556). We also noted that expression of the reporter gene is not observed following microinjection of the germinal vesicle of the fully grown oocyte. In the present study, we examined expression of this reporter gene during oocyte growth, as well as the nuclear concentration of two transcription factors, Sp1 and the TATA box-binding protein, TBP, during oocyte growth and the first cell cycle. The extent of reporter gene expression decreases during oocyte growth and this decrease correlates with the decrease in nuclear concentration of Sp1, as determined by confocal immunofluorescent microscopy. In addition, results of immunoblotting experiments also indicate a similar decrease in the total concentration of Sp1 during oocyte growth. The nuclear concentration of TBP also decreases during oocyte growth, as determined by confocal immunofluorescent microscopy. Following fertilization, the pronuclear concentration of these two transcription factors increases in a time-dependent fashion and the concentration of each is greater in the male pronucleus as compared to the female pronucleus. For each pronucleus and for each transcription factor, this increase in nuclear concentration is inhibited by aphidicolin, which inhibits DNA synthesis. Last, the increase in nuclear concentration of these two proteins observed between the 1-cell and 2-cell stages does not require transcription or cytokinesis. PMID: 7925035 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 69: Genomics. 1994 Jul 1;22(1):94-100. Physical mapping at 6q27 of the locus for the TATA box-binding protein, the DNA-binding subunit of TFIID and a component of SL1 and TFIIIB, strongly suggests that it is single copy in the human genome. Purrello M, Di Pietro C, Mirabile E, Rapisarda A, Rimini R, Tine A, Pavone L, Motta S, Grzeschik KH, Sichel G. Istituto di Biologia generale, Universita di Catania, Italy. The TATA box-binding protein (TBP) has a fundamental role in eukaryotic cell metabolism, since it is necessary for transcription of class I, class II, and class III genes; in fact, TBP is the DNA-binding subunit of TFIID and a component of SL1 and TFIIIB. Contrary to the previously hypothesized existence of a family of genes coding for DNA-binding proteins highly related to TBP, our experiments show that the segment coding for the evolutionarily well-conserved carboxyl-terminal domain, involved in DNA binding, is unique; accordingly, we conclude that the TBP locus itself, which we have localized to 6q27, is single copy in the human genome. On the other hand, a cDNA fragment coding for the evolutionarily variable amino-terminal domain detects multiple cross-hybridizing sequences in the genome of higher eukaryotes. We suggest that the common motif is represented by the long string of glutamine codons, which characterizes the amino-terminal segment of human TBP: in fact, other proteins involved in transcription, such as TAF II 110, Sp1, and some homeobox proteins, are known to contain glutamine-rich segments. PMID: 7959796 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 70: Mol Cell Biol. 1994 Mar;14(3):1582-93. Species-specific interaction of the glutamine-rich activation domains of Sp1 with the TATA box-binding protein. Emili A, Greenblatt J, Ingles CJ. Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada. We have used protein-blotting and protein affinity chromatography to demonstrate that each of the two glutamine-rich activation domains of the human transcription factor Sp1 can bind specifically and directly to the C-terminal evolutionarily conserved domain of the human TATA box-binding protein (TBP). These activation domains of Sp1 also bind directly to Drosophila TBP but bind much less strongly to TBP from the yeast Saccharomyces cerevisiae. The abilities of the Sp1 activation domains to interact directly with the TBPs of various species correlate well with their abilities to activate transcription in extracts derived from the same species. We also show that a glutamine-rich transcriptional activating region of the Drosophila protein Antennapedia binds directly to TBP in a species-specific manner that reflects its ability to activate transcription in vivo. These results support the notion that TBP is a direct and important target of glutamine-rich transcriptional activators. PMID: 8114696 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 71: Nucleic Acids Res. 1994 Feb 25;22(4):669-77. Sp1 is essential for both enhancer-mediated and basal activation of the TATA-less human adenosine deaminase promoter. Dusing MR, Wiginton DA. Department of Pediatrics, University of Cincinnati College of Medicine, OH. Tissue-specific expression of the human adenosine deaminase (ADA) gene is mediated by transcriptional activation over a thousand-fold range. Cis-regulatory regions responsible for high and basal levels of activation include an enhancer and the proximal promoter region. While analyses of the T-cell specific enhancer have been carried out, detailed studies of the the promoter region or promoter-enhancer interactions have not. Examination of the promoter region by homology searches revealed six putative Sp1 binding sites. DNase I footprinting showed that Sp1 is able to bind these sites. Deletion analysis indicated that the proximal Sp1 site is required for activation of a reporter gene to detectable levels and that the more distal Sp1 sites further activate the level of expression. Inclusion of an ADA enhancer-containing fragment in these deletion constructions demonstrated that Sp1 sites are also essential for enhancer function. Apparently Sp1 controls not only low level expression but is also an integral part of the mechanism by which the enhancer achieves high level ADA expression. Mutagenesis of a potential TBP binding site at base pairs -21 to -26 decreased activity only two-fold indicating that it is not essential for transcriptional activation or enhancement. PMID: 8127716 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 72: Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):192-6. A glutamine-rich hydrophobic patch in transcription factor Sp1 contacts the dTAFII110 component of the Drosophila TFIID complex and mediates transcriptional activation. Gill G, Pascal E, Tseng ZH, Tjian R. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720. Activation of transcription by the promoter-specific factor Sp1 requires coactivators that are tightly associated with the TATA-box-binding protein (TBP) in the TFIID complex. Recent work has shown that the two glutamine-rich activation domains of Sp1, A and B, can interact with at least one component of this complex, the TBP-associated factor dTAFII110. Here we report the mapping of a region of Sp1 with alternating glutamine and hydrophobic residues which is required for the interaction with dTAFII110 and is important for mediating transcriptional activation. Substitution of bulky hydrophobic residues within this region decreased both interaction with dTAFII110 and transcriptional activation in Drosophila cells. In contrast, mutation of glutamine residues in this region had no effect. Thus, the strength of the Sp1-TAF interaction correlates with the potency of Sp1 as a transcriptional activator, indicating that this activator-TAF interaction is an important part of the mechanism of transcriptional activation. Sequence comparison of three activation domains shown to bind dTAFII110 suggests that different activators that utilize dTAFII110 as a coactivator may share common sequence features that we have determined to be important for the Sp1-dTAFII110 interaction. PMID: 8278363 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 73: EMBO J. 1994 Jan 1;13(1):147-57. Cooperativity in vivo between the E2 transactivator and the TATA box binding protein depends on core promoter structure. Ham J, Steger G, Yaniv M. Unite des Virus Oncogenes, VA 1644 du CNRS, Departement des Biotechnologies, Institut Pasteur, Paris, France. The E2 transactivator protein of bovine papillomavirus 1 (BPV-1) can strongly stimulate complex promoters such as that of the herpes simplex virus thymidine kinase gene but does not efficiently activate minimal promoters that only contain E2 binding sites and a TATA box. Here we show that overexpression of the human, but not yeast, TATA box binding protein (TBP) in transfection experiments overcomes this block and enables E2 to activate a minimal TATA box-containing promoter. This suggests that recruitment of the TFIID complex to such promoters is normally a rate limiting step for transcriptional activation by E2 in vivo. In contrast, minimal promoters that contain an initiator element in addition to a TATA box are efficiently activated by E2 on its own and this activation is only moderately enhanced by TBP overexpression. In such E2-responsive promoters the TATA box or initiator can be functionally replaced by SP1 binding sites. Both the initiator binding protein, TFII-I, and SP1 have been found to interact physically with components of the TFIID complex. Since either TBP overexpression or the presence of an initiator or SP1 binding sites can increase activation by E2, it seems likely that the principal role of the E2 activation domain is to affect a step in the formation of the transcription initiation complex that occurs after TFIID has bound to the promoter. Sequential action of transcription factors, such as TFII-I, SP1 and E2, may be one type of mechanism underlying the widely observed phenomenon of transcriptional synergy. PMID: 8306958 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 74: Nucleic Acids Res. 1993 Dec 11;21(24):5609-15. C-terminal domain (CTD) of RNA-polymerase II and N-terminal segment of the human TATA binding protein (TBP) can mediate remote and proximal transcriptional activation, respectively. Seipel K, Georgiev O, Gerber HP, Schaffner W. Institut fur Molekularbiologie II, Universitat Zurich, Switzerland. Activation domains of mammalian transcription factors can be subdivided into at least two functional classes. One, exemplified by the glutamine-rich activation domains of Oct and Sp1 factors, mediates transcriptional activation only from a proximal promoter position, and in response to an enhancer. The other, exemplified by the 'acidic' domain of the viral activator VP16, has the ability to activate from remote enhancer as well as from proximal promoter positions. Here we report that two proteins of the basal transcription apparatus also contain activation domains whose stimulatory effect can be detected in fusion proteins containing the GAL4 DNA binding domain. The human TATA-binding protein (TBP) contains at its N-terminus a domain with typical 'promoter' activity. We propose that the TBP N-terminal region acts as an auxiliary activation domain which reinforces the activity of other promoter-bound factors. The largest subunit of RNA polymerase II contains at its C-terminus a conserved heptad repeat structure (CTD). Both natural and synthetic CTD consensus repeats fused to GAL4 can activate transcription from remote positions like a typical enhancer-active domain. Accordingly we propose that the RNA polymerase II large subunit contains a 'portable' domain for transcriptional activation which may synergize with the activation domains of enhancer-bound transcription factors. PMID: 8284205 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 75: Cell. 1993 Nov 5;75(3):519-30. Drosophila TAFII40 interacts with both a VP16 activation domain and the basal transcription factor TFIIB. Goodrich JA, Hoey T, Thut CJ, Admon A, Tjian R. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720. Enhancement of RNA polymerase II transcription by the viral transactivator VP16 requires the TFIID complex, which consists of the TATA-binding protein (TBP) and TBP-associated factors (TAFs). Here we report the molecular cloning, expression, and biochemical characterization of Drosophila TAFII40 (dTAFII40), a subunit of TFIID. In vitro protein-protein interaction assays revealed direct binding between dTAFII40 and a 39 amino acid VP16 activation domain. In addition, affinity chromatography indicated a direct binding of the basal factor TFIIB to immobilized dTAFII40. Since VP16 also binds TFIIB, our results suggest a ternary interaction among an activator, a coactivator, and a basal transcription factor. Antibodies directed against dTAFII40 inhibited activation by GAL4-VP16 without affecting basal transcription. These results, taken together with previous studies of Sp1 and dTAFII110, establish that different activators interact with distinct TAFs in the TFIID complex and that TAFs can contact both activators and basal factors. PMID: 8221891 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 76: Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9528-32. Herpes simplex virus infected cell polypeptide 4 preferentially represses Sp1-activated over basal transcription from its own promoter. Gu B, Rivera-Gonzalez R, Smith CA, DeLuca NA. Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine 15261. Herpes simplex virus type 1 infected cell polypeptide 4 (HSV-1 ICP4) is a multifunctional phosphoprotein that is essential for viral infection. It is both a repressor and an activator of viral gene expression depending upon the promoter. ICP4 represses transcription from its own promoter. In the present study, we used general transcription factors from HeLa cell nuclear extracts, recombinant TATA binding protein (TBP) and TFIIB, and the transcriptional activator Sp1 to reconstitute in vitro transcription for the ICP4 promoter and to examine the effects of purified ICP4 on transcription. ICP4 was able to effectively repress Sp1-induced transcription from ICP4 promoter templates that contain one or multiple Sp1 binding sites. The observed inhibition required the ICP4 binding site that spans the transcription initiation site. ICP4 did not inhibit basal transcription as inferred by its inability to inhibit transcription when (i) Sp1 was not included in transcription reactions, (ii) the templates contained no Sp1 binding sites, and (iii) TBP was used in place of TFIID in the reactions. The in vitro observations were consistent with the behavior of the same constructs expressed in cells from the herpes simplex virus type 1 genome. DNase I footprinting experiments revealed that ICP4 could co-occupy the ICP4 promoter region with TBP-TFIIB, indicating that ICP4 does not necessarily exclude these factors from binding to the TATA region. The data suggest that the repressive effects of ICP4 observed in this study result from ICP4 interfering with the interactions contributing to Sp1-induced transcription. PMID: 8415735 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 77: Mol Cell Biol. 1993 Jul;13(7):3841-9. Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence. Zenzie-Gregory B, Khachi A, Garraway IP, Smale ST. Howard Hughes Medical Institute, UCLA School of Medicine 90024-1662. Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex. PMID: 8321191 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 78: Nature. 1993 May 13;363(6425):176-9. The dTAFII80 subunit of Drosophila TFIID contains beta-transducin repeats. Dynlacht BD, Weinzierl RO, Admon A, Tjian R. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720. A key component of the RNA polymerase II transcriptional apparatus, TFIID, is a multi-protein complex containing the TATA box-binding protein (TBP) and at least seven tightly associated factors (TAFs). Although the functions of most TFIID subunits are unknown, it is clear that TAFs are not necessary for basal activity but that one or more are required for regulated transcription, and so behave as coactivators. The presence of multiple subunits indicates that there is an intricate assembly process and that TAFs may be responsible for other activities. We have described the properties of the subunit dTAFII110, which can interact directly with the transcriptional activator Sp1 (ref. 5). In addition, the largest subunit, dTAFII250, binds directly to TBP and links other TAFs to the complex. Here we describe the cloning, expression and partial characterization of the Drosophila TAF of M(r) 80,000, dTAFII80. Sequence analysis reveals that dTAFII80 contains several copies of the WD40 (beta-transducin) repeat. Moreover, dTAFII80 shares extended sequence similarity with an Arabidopsis gene, COP1, which encodes a putative transcription factor that is though to regulate development. We have expressed recombinant dTAFII80 and begun to characterize its interaction with other members of the TFIID complex. Purified recombinant dTAFII80 is unable to bind TBP directly or to interact strongly with the C-terminal domain of dTAFII250 (delta N250). Instead, dTAFII80 is only able to recognize and interact with a higher-order complex containing TBP, delta N250, 110 and 60. These findings suggest the formation of TFIID may require an ordered assembly of the TAFs, some of which bind directly to TBP and others that are tethered to the complex as a result of specific TAF/TAF interactions. PMID: 8483503 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 79: Nature. 1993 Apr 8;362(6420):511-7. Largest subunit of Drosophila transcription factor IID directs assembly of a complex containing TBP and a coactivator. Weinzierl RO, Dynlacht BD, Tjian R. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720. The TFIID complex consists of the TATA-binding protein (TBP) and associated factors (TAFs) serving to mediate transcriptional activation by promoter-specific regulators. Here we report the cloning of Drosophila TAFII250 and the assembly of a partial complex containing recombinant TBP, TAFII110 and the C-terminal domain of TAFII250. This triple complex supports Sp1 activation and reveals specific interactions between TAFII250, TBP and TAFII110. PMID: 8464492 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 80: J Virol. 1992 Sep;66(9):5453-63. Substitution of a TATA box from a herpes simplex virus late gene in the viral thymidine kinase promoter alters ICP4 inducibility but not temporal expression. Imbalzano AN, DeLuca NA. Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115. The role of cis-acting promoter elements associated with herpes simplex virus type 1 (HSV-1) early and late genes was evaluated during productive infection with regard to activation of gene expression by the HSV-1 transactivator ICP4 and control of temporal regulation. A set of recombinant viruses was constructed such that expression of an HSV-1 early gene, thymidine kinase (tk), was placed under the control of either the tk TATA box or the TATA box from the late gene, glycoprotein C (gC), in the presence or absence of the upstream Sp1 and CCAAT sites normally found in the tk promoter. The presence of Sp1 sites in the promoter or replacement of the tk TATA box with the gC TATA box resulted in a decreased activation of tk mRNA expression by ICP4. Substitution of the A + T-rich region from the gC TATA box in the context of the remainder of the surrounding tk sequences resulted in a promoter that bound recombinant TATA-binding protein (TBP) better at lower concentrations than the wild-type tk promoter did. These results indicate that tk promoters that are better able to utilize TBP are less responsive to ICP4 activation and suggest that activation by ICP4 involves the general transcription factors that interact with TBP or TBP itself. Additionally, all of the viruses expressed tk at early times postinfection, indicating that cis-acting promoter elements that control the level of expression of HSV-1 early and late genes do not determine temporal regulation. PMID: 1323706 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 81: Genes Dev. 1991 Dec;5(12A):2212-24. Coactivators for a proline-rich activator purified from the multisubunit human TFIID complex. Tanese N, Pugh BF, Tjian R. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720. The mechanisms of transcriptional activation directed by sequence-specific regulators is central to understanding gene regulation. Here, we report the isolation of coactivators responsible for mediating transcriptional activation by Gal4-Pro, a hybrid regulator containing the proline-rich activation domain of human CTF/NFI. Chromatographic studies indicate that endogenous human TFIID consists of a multisubunit complex containing the TATA-binding protein (TBP), coactivators, and other associated factors. A fraction containing the coactivator activity was separated from the endogenous TBP after disrupting the tightly associated complex with urea. The urea-purified TBP was active for basal level transcription but no longer could support activation by Gal4-Pro. However, when the two separated components were added together, activated levels of transcription were restored in the presence of Gal4-Pro. Immunoaffinity purification of the TFIID complex identifies several polypeptides specifically associated with the endogenous TBP, some or all of which function as coactivators when reconstituted with Gal4-Pro. The isolated coactivators also mediate activation by a chimeric glutamine-rich activator derived from Sp1 but not the Gal4-VP16 activator, suggesting distinct factor requirements for different types of transcriptional regulators. PMID: 1748279 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 82: Genes Dev. 1991 Nov;5(11):1935-45. Transcription from a TATA-less promoter requires a multisubunit TFIID complex. Pugh BF, Tjian R. Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720. In eukaryotes, the TATA box-binding protein (TBP) is responsible for nucleating assembly of the transcription initiation machinery. Here, we report that a TFIID complex containing TBP is essential for transcription even at a promoter that lacks a TATA box. Immunopurification of TFIID reveals that the active species in reconstituting TATA-less transcription is a multisubunit complex consisting of TBP and many TBP-associated factors (TAFs). PMID: 1657708 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------