Eukaryotic Transcription Gene Regulation

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Improved ecdysone receptor-based inducible gene regulation system Subba R. Palli 1 , Mariana Z. Kapitskaya 2 , Mohan B. Kumar 2 and Dean E. Cress 2 1 Department of Entomology, College of Agriculture, University of Kentucky, KY, USA; 2 RHeoGene LLC, Spring House, PA, USA To develop an ecdysone receptor (EcR)-based inducible gene regulation system, several constructs were prepared by fusing DEF domains of Choristoneura fumiferana EcR (CfEcR), C. fumiferana ultraspiracle (CfUSP), Mus muscu- lus retinoid X receptor (MmRXR) to either GAL4 DNA binding domain (DBD) or VP16 activation domain. These constructs were tested in mammalian cells to evaluate their ability to transactivate luciferase gene placed under the control of GAL4 response elements and synthetic TATAA promoter. A two-hybrid format switch, where GAL4 DBD was fused to CfEcR (DEF) and VP16 AD was fused to MmRXR (EF) was found to be the best combination. It had the lowest background levels of reporter gene activity in the absence of a ligand and the highest level of reporter gene activity in the presence of a ligand. Both induction and turn- off responses were fast. A 16-fold induction was observed within 3 h of ligand addition and increased to 8942-fold by 48 h after the addition of ligand. Withdrawal of the ligand resulted in 50% and 80% reduction in reporter gene activity by 12 h and 24 h, respectively. Keywords: gene switch; ponasterone A; receptors; EcR; RXR. Twenty hydroxyecdysone (20E) is a steroid hormone that regulates molting, metamorphosis, reproduction and vari- ous other developmental processes in insects. Ecdysone functions through a heterodimeric receptor complex com- prised of ecdysone receptor (EcR) and ultraspiracle (USP). Both EcR and USP cDNAs have been cloned from Drosophila melanogaster and several other insects [1] and were shown to be members of the steroid hormone receptor superfamily. Members of this superfamily are characterized by the presence of five modular domains, A/B (transacti- vation), C (DNA binding/heterodimerization), D (hinge, heterodimerization), E (ligand binding, heterodimerization, transactivation) and F (transactivation). Crystallographic studies on the E domain structures of several nuclear receptors showed a conserved fold composed of 11 helices (H1 and H3–H12) and two short strands (s1 and s2) [2]. Recently, the crystal structure of USP was solved by two groups [3,4], both structures showed a long H1-H3 loop and an insert between H5 and H6. These structures appear to lock USP in an inactive conformation by displacing helix 12 from agonist conformation. In both crystal structures USP had a large hydrophobic cavity, which contained phos- pholipid ligands. The crystal structure of the EcR has yet to be determined; however, homology models for CtEcR (Chironomus tentans EcR) [5], and CfEcR (Choristoneura fumiferana EcR) [6] have been generated [7,8]. Ecdysone receptors are found in insects and other related invertebrates [1]. Ecdysteroids and related compounds have been identified in plants, insects and other related inverte- brates. EcR and its ligands are not detected in vertebrates such as humans, therefore they are very good candidates for developing gene regulation systems for use in vertebrates. Insect EcR can heterodimerize with retinoid X receptor (RXR) and transactivate genes that are placed under the control of ecdysone response elements (EcRE) in various cellular backgrounds including mammalian cells. The EcR- based gene switch is being developed for use in various applications including gene therapy, expression of toxic proteins in cell lines as well as for cell-based drug discovery assays [9–17]. After initial reports [18,19] on the function of EcR as an ecdysteroid dependent transcription factor in cultured mammalian cells, No et al. [20] used D. melanogaster EcR (DmEcR) and human RXRa to develop Eukaryotic Transcription Gene Regulation Eukaryotic Transcription Gene Regulation Bởi: OpenStaxCollege Like prokaryotic cells, the transcription of genes in eukaryotes requires the actions of an RNA polymerase to bind to a sequence upstream of a gene to initiate transcription However, unlike prokaryotic cells, the eukaryotic RNA polymerase requires other proteins, or transcription factors, to facilitate transcription initiation Transcription factors are proteins that bind to the promoter sequence and other regulatory sequences to control the transcription of the target gene RNA polymerase by itself cannot initiate transcription in eukaryotic cells Transcription factors must bind to the promoter region first and recruit RNA polymerase to the site for transcription to be established Link to Learning View the process of transcription—the making of RNA from a DNA template—at this site The Promoter and the Transcription Machinery Genes are organized to make the control of gene expression easier The promoter region is immediately upstream of the coding sequence This region can be short (only a few nucleotides in length) or quite long (hundreds of nucleotides long) The longer the promoter, the more available space for proteins to bind This also adds more control to the transcription process The length of the promoter is gene-specific and can differ dramatically between genes Consequently, the level of control of gene expression can also differ quite dramatically between genes The purpose of the promoter is to bind transcription factors that control the initiation of transcription 1/4 Eukaryotic Transcription Gene Regulation Within the promoter region, just upstream of the transcriptional start site, resides the TATA box This box is simply a repeat of thymine and adenine dinucleotides (literally, TATA repeats) RNA polymerase binds to the transcription initiation complex, allowing transcription to occur To initiate transcription, a transcription factor (TFIID) is the first to bind to the TATA box Binding of TFIID recruits other transcription factors, including TFIIB, TFIIE, TFIIF, and TFIIH to the TATA box Once this complex is assembled, RNA polymerase can bind to its upstream sequence When bound along with the transcription factors, RNA polymerase is phosphorylated This releases part of the protein from the DNA to activate the transcription initiation complex and places RNA polymerase in the correct orientation to begin transcription; DNA-bending protein brings the enhancer, which can be quite a distance from the gene, in contact with transcription factors and mediator proteins ([link]) An enhancer is a DNA sequence that promotes transcription Each enhancer is made up of short DNA sequences called distal control elements Activators bound to the distal control elements interact with mediator proteins and transcription factors Two different genes may have the same promoter but different distal control elements, enabling differential gene expression In addition to the general transcription factors, other transcription factors can bind to the promoter to regulate gene transcription These transcription factors bind to the promoters of a specific set of genes They are not general transcription factors that bind to every promoter complex, but are recruited to a specific sequence on the promoter of a specific gene There are hundreds of transcription factors in a cell that each bind specifically to a particular DNA sequence motif When transcription factors bind to the promoter just upstream of the encoded gene, it is referred to as a cis-acting element, 2/4 Eukaryotic Transcription Gene Regulation because it is on the same chromosome just next to the gene The region that a particular transcription factor binds to is called the transcription factor binding site Transcription factors respond to environmental stimuli that cause the proteins to find their binding sites and initiate transcription of the gene that is needed Enhancers and Transcription In some eukaryotic genes, there are regions that help increase or enhance transcription These regions, called enhancers, are not necessarily close to the genes they enhance They can be located upstream of a gene, within the coding region of the gene, downstream of a gene, or may be thousands of nucleotides away Enhancer regions are binding sequences, or sites, for transcription factors When a DNA-bending protein binds, the shape of the DNA changes ([link]) This shape change allows for the interaction of the activators bound to the enhancers with the transcription factors bound to the promoter region and the RNA polymerase Whereas DNA is generally depicted as a straight line in two dimensions, it is actually a three-dimensional object Therefore, a nucleotide sequence thousands of nucleotides away can fold over and interact with a specific promoter Turning Genes Off: Transcriptional Repressors Like prokaryotic cells, eukaryotic cells also have mechanisms to prevent transcription ...Genome Biology 2007, 8:R181 Open Access 2007Heet al.Volume 8, Issue 9, Article R181 Research Dynamic cumulative activity of transcription factors as a mechanism of quantitative gene regulation Feng He * , Jan Buer †‡ , An-Ping Zeng §¶ and Rudi Balling * Addresses: * Biological Systems Analysis Group, HZI- Helmholtz Centre for Infection Research, Inhoffenstrasse, D-38124 Braunschweig, Germany. † Mucosal Immunity Group, HZI- Helmholtz Centre for Infection Research, Inhoffenstrasse, D-38124 Braunschweig, Germany. ‡ Institute of Medical Microbiology, Hannover Medical School (MHH), D-30625 Hannover, Germany. § Systems Biology Group, HZI- Helmholtz Centre for Infection Research, Inhoffenstrasse, D-38124 Braunschweig, Germany. ¶ Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, Denickerstrasse, D-21073 Hamburg, Germany. Correspondence: Rudi Balling. Email: Rudi.Balling@helmholtz-hzi.de © 2007 He et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dynamic cumulative transcriptional regulation<p>By combining information on the yeast transcription network and high-resolution time-series data with a series of factors, support is provided for the concept that dynamic cumulative regulation is a major principle of quantitative transcriptional control.</p> Abstract Background: The regulation of genes in multicellular organisms is generally achieved through the combinatorial activity of different transcription factors. However, the quantitative mechanisms of how a combination of transcription factors controls the expression of their target genes remain unknown. Results: By using the information on the yeast transcription network and high-resolution time- series data, the combinatorial expression profiles of regulators that best correlate with the expression of their target genes are identified. We demonstrate that a number of factors, particularly time-shifts among the different regulators as well as conversion efficiencies of transcription factor mRNAs into functional binding regulators, play a key role in the quantification of target gene expression. By quantifying and integrating these factors, we have found a highly significant correlation between the combinatorial time-series expression profile of regulators and their target gene expression in 67.1% of the 161 known yeast three-regulator motifs and in 32.9% of 544 two-regulator motifs. For network motifs involved in the cell cycle, these percentages are much higher. Furthermore, the results have been verified with a high consistency in a second independent set of time-series data. Additional support comes from the finding that a high percentage of motifs again show a significant correlation in time-series data from stress-response studies. Conclusion: Our data strongly support the concept that dynamic cumulative regulation is a major principle of quantitative transcriptional control. The proposed concept might also apply to other organisms and could be relevant for a wide range of biotechnological applications in which quantitative gene regulation plays a role. Published: 4 September 2007 Genome Biology 2007, 8:R181 (doi:10.1186/gb-2007-8-9-r181) Received: 24 April 2007 Revised: 22 August 2007 Accepted: 4 September 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/9/R181 R181.2 Genome Biology 2007, Volume 8, Issue 9, Article R181 He et al. http://genomebiology.com/2007/8/9/R181 Genome Biology 2007, 8:R181 Background One of the important elements of gene regulation is mediated by the binding of transcription factors to specific binding sites of promoters or other gene regulatory control regions. In Genome Biology 2005, 6:R87 comment reviews reports deposited research refereed research interactions information Open Access 2005Granek and ClarkeVolume 6, Issue 10, Article R87 Method Explicit equilibrium modeling of transcription-factor binding and gene regulation Joshua A Granek *† and Neil D Clarke *‡ Addresses: * Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, North Wolfe Street, Baltimore, MD 21205, USA. † National Evolutionary Synthesis Center, Broad Street, Durham, NC 27705, USA. ‡ Genome Institute of Singapore, Biopolis Street, Singapore 138672, Republic of Singapore. Correspondence: Neil D Clarke. E-mail: nclarke@jhmi.edu © 2005 Granek and Clarke; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Explicit equilibrium modeling of transcription-factor binding and gene regulation<p>A computational model, GOMER, is presented that predicts transcription-factor binding and incorporates effects of cooperativity and competition.</p> Abstract We have developed a computational model that predicts the probability of transcription factor binding to any site in the genome. GOMER (generalizable occupancy model of expression regulation) calculates binding probabilities on the basis of position weight matrices, and incorporates the effects of cooperativity and competition by explicit calculation of coupled binding equilibria. GOMER can be used to test hypotheses regarding gene regulation that build upon this physically principled prediction of protein-DNA binding. Background Transcription is regulated by the binding of proteins to spe- cific DNA sequences. Until recently, binding and regulation could only be studied at the level of individual genes, but they can now be studied as a complex system due to the availability of genome-wide data on expression and transcription factor binding. Computational models are needed, however, to eval- uate co-regulated genes and the sequence motifs associated with them. A general strategy for testing the relevance of a DNA binding motif to gene regulation is to quantify the association of the motif with co-regulated genes. This can be done by comparing the regulatory sequences of co-regulated genes with the regu- latory sequences of all other genes [1-4]. One simple test is to score for the occurrence of a consensus site within a pre- scribed distance 5' to the start of transcription. If the fraction of regulated genes with a consensus site is significantly larger than the fraction of unregulated genes, as it often is, then the test has some predictive power [1,5-7]. As with all statistical tests, there is a model implicit in this test: in this case, the implicit model is that gene regulation is mediated by a single consensus binding site. There are problems with such a simple model. First, the use of consensus binding sites, even if degenerate, underesti- mates the importance of motifs that resemble the consensus but do not match it [8]. At the same time, degenerate consen- sus sites fail to distinguish among motifs that match the con- sensus even if the motifs that match differ in affinity. Second, regulated genes often contain more than one binding site for a given factor, so scoring based on a single site (or any other threshold number of sites) is arbitrary. Third, the binding of a factor is typically affected by cooperative and competitive interactions with other proteins, so binding sites for those other proteins may need to be considered. Fourth, gene expression can be affected by the location, orientation and spacing of bound transcription factors. Therefore, to be real- istic, a model for gene regulation should use to full advantage an accurate representation of The splicing factor ASF/SF2 is associated with TIA-1-related/ TIA-1-containing ribonucleoproteic complexes and contributes to post-transcriptional repression of gene expression Nathalie Delestienne 1 , Corinne Wauquier 1 , Romuald Soin 1 , Jean-Franc¸ois Dierick 2, *, Cyril Gueydan 1, and Ve ´ ronique Kruys 1, 1 Laboratoire de Biologie Mole ´ culaire du Ge ` ne, Faculte ´ des Sciences, Universite ´ Libre de Bruxelles, Gosselies, Belgium 2 Biovalle ´ e, Proteomics Unit, Charleroi, Belgium Keywords AU-rich elements; hnRNP, heterogenous nuclear ribonucleoprotein; ribonucleoprotein complexes; RNA metabolism; RNA-binding proteins; stress granules Correspondence V. Kruys, Laboratoire de Biologie Mole ´ culaire du Ge ` ne, Institut de Biologie et de Me ´ decine Mole ´ culaires, Universite ´ Libre de Bruxelles, 12 rue des Profs. Jeener et Brachet, 6041 Gosselies, Belgium Fax: +32 2 6509800 Tel: +32 2 6509804 E-mail: vkruys@ulb.ac.be *Present address GSK Biologicals, Wavre, Belgium  These authors contributed equally to this work (Received 10 January 2010, revised 10 March 2010, accepted 25 March 2010) doi:10.1111/j.1742-4658.2010.07664.x TIA-1-related (TIAR) protein is a shuttling RNA-binding protein impli- cated in several steps of RNA metabolism. In the nucleus, TIAR contrib- utes to alternative splicing events, whereas, in the cytoplasm, it acts as a translational repressor on specific transcripts such as adenine and uridine- rich element-containing mRNAs. In addition, TIAR is involved in the general translational arrest observed in cells exposed to environmental stress. This activity is encountered by the ability of TIAR to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules. To elucidate these mechanisms of translational repression, we characterized TIAR-containing complexes by tandem affinity purification followed by MS. Amongst the identified proteins, we found the splicing factor ASF ⁄ SF2, which is also present in TIA-1 protein complexes. We show that, although mostly confined in the nuclei of normal cells, ASF ⁄ SF2 migrates into stress granules upon environmental stress. The migration of ASF ⁄ SF2 into stress granules is strictly determined both by its shuttling properties and its RNA-binding capacity. Our data also indi- cate that ASF ⁄ SF2 down-regulates the expression of a reporter mRNA carrying adenine and uridine-rich elements within its 3¢ UTR. Moreover, tethering of ASF ⁄ SF2 to a reporter transcript strongly reduces mRNA translation and stability. These results indicate that ASF ⁄ SF2 and TIA proteins cooperate in the regulation of mRNA metabolism in normal cells and in cells having to overcome environmental stress conditions. In addi- tion, the present study provides new insights into the cytoplasmic function of ASF ⁄ SF2 and highlights mechanisms by which RNA-binding proteins regulate the diverse steps of RNA metabolism by subcellular relocalization upon extracellular stimuli. Structured digital abstract l MINT-7715509: ASF ⁄ SF2 (uniprotkb:Q6PDM2)andTIAR (uniprotkb:P70318) colocalize (MI:0403) by fluorescence microscopy ( MI:0416) Abbreviations ARE, adenine and uridine-rich element; CBB, calmodulin binding buffer; CP, coat protein; FITC, fluorescein isothiocyanate; Fluc, firefly luciferase; HA, haemagglutinin; IP, immunoprecipitation; NLS, nuclear localization signal; NPc, nucleoplasmin core domain; Rluc, Renilla luciferase; RRM, RNA recognition motif; RS, arginine-serine; SG, stress granule; SR, serine-arginine; TAP, tandem affinity purification; TIAR, TIA-1-related. 2496 FEBS Journal 277 Eukaryotic Post-transcriptional Gene Regulation Eukaryotic Posttranscriptional Gene Regulation Bởi: OpenStaxCollege RNA is transcribed, but must be processed into a mature form before translation can begin This processing after an RNA molecule has been transcribed, but before it is translated into a protein, is called Improved ecdysone receptor-based inducible gene regulation system Subba R. Palli 1 , Mariana Z. Kapitskaya 2 , Mohan B. Kumar 2 and Dean E. Cress 2 1 Department of Entomology, College of Agriculture, University of Kentucky, KY, USA; 2 RHeoGene LLC, Spring House, PA, USA To develop an ecdysone receptor (EcR)-based inducible gene regulation system, several constructs were prepared by fusing DEF domains of Choristoneura fumiferana EcR (CfEcR), C. fumiferana ultraspiracle (CfUSP), Mus muscu- lus retinoid X receptor (MmRXR) to either GAL4 DNA binding domain (DBD) or VP16 activation domain. These constructs were tested in mammalian cells to evaluate their ability to transactivate luciferase gene placed under the control of GAL4 response elements and synthetic TATAA promoter. A two-hybrid format switch, where GAL4 DBD was fused to CfEcR (DEF) and VP16 AD was fused to MmRXR (EF) was found to be the best combination. It had the lowest background levels of reporter gene activity in the absence of a ligand and the highest level of reporter gene activity in the presence of a ligand. Both induction and turn- off responses were fast. A 16-fold induction was observed within 3 h of ligand addition and increased to 8942-fold by 48 h after the addition of ligand. Withdrawal of the ligand resulted in 50% and 80% reduction in reporter gene activity by 12 h and 24 h, respectively. Keywords: gene switch; ponasterone A; receptors; EcR; RXR. Twenty hydroxyecdysone (20E) is a steroid hormone that regulates molting, metamorphosis, reproduction and vari- ous other developmental processes in insects. Ecdysone functions through a heterodimeric receptor complex com- prised of ecdysone receptor (EcR) and ultraspiracle (USP). Both EcR and USP cDNAs have been cloned from Drosophila melanogaster and several other insects [1] and were shown to be members of the steroid hormone receptor superfamily. Members of this superfamily are characterized by the presence of five modular domains, A/B (transacti- vation), C (DNA binding/heterodimerization), D (hinge, heterodimerization), E (ligand binding, heterodimerization, transactivation) and F (transactivation). Crystallographic studies on the E domain structures of several nuclear receptors showed a conserved fold composed of 11 helices (H1 and H3–H12) and two short strands (s1 and s2) [2]. Recently, the crystal structure of USP was solved by two groups [3,4], both structures showed a long H1-H3 loop and an insert between H5 and H6. These structures appear to lock USP in an inactive conformation by displacing helix 12 from agonist conformation. In both crystal structures USP had a large hydrophobic cavity, which contained phos- pholipid ligands. The crystal structure of the EcR has yet to be determined; however, homology models for CtEcR (Chironomus tentans EcR) [5], and CfEcR (Choristoneura fumiferana EcR) [6] have been generated [7,8]. Ecdysone receptors are found in insects and other related invertebrates [1]. Ecdysteroids and related compounds have been identified in plants, insects and other related inverte- brates. EcR and its ligands are not detected in vertebrates such as humans, therefore they are very good candidates for developing gene regulation systems for use in vertebrates. Insect EcR can heterodimerize with retinoid X receptor (RXR) and transactivate genes that are placed under the control of ecdysone response elements (EcRE) in various cellular backgrounds including mammalian cells. The EcR- based gene switch is being developed for use in various applications including gene therapy, expression of toxic proteins in cell lines as well as for cell-based drug discovery assays [9–17]. After initial reports [18,19] on the function of EcR as an ecdysteroid dependent transcription factor in cultured mammalian cells, No et al. [20] used D. melanogaster EcR (DmEcR) and human RXRa to develop Eukaryotic Epigenetic Gene ... factors, other transcription factors can bind to the promoter to regulate gene transcription These transcription factors bind to the promoters of a specific set of genes They are not general transcription. .. encoded gene, it is referred to as a cis-acting element, 2/4 Eukaryotic Transcription Gene Regulation because it is on the same chromosome just next to the gene The region that a particular transcription. .. prevent transcription Review Questions The binding of is required for transcription to start a protein DNA polymerase 3/4 Eukaryotic Transcription Gene Regulation RNA polymerase a transcription