Eukaryotic Transcription

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Int. J. Med. Sci. 2007, 4 28International Journal of Medical Sciences ISSN 1449-1907 www.medsci.org 2007 4(1):28-35 © Ivyspring International Publisher. All rights reserved Research Paper Functional genomics analysis of low concentration of ethanol in human hepatocellular carcinoma (HepG2) cells. Role of genes involved in transcriptional and translational processes Francisco Castaneda 1, Sigrid Rosin-Steiner 1 and Klaus Jung 2 3 1. Laboratory for Molecular Pathobiochemistry and Clinical Research, Max Planck Institute of Molecular Physiology, Dortmund, Germany; 2. Department of Statistics, University of Dortmund, D-44221 Dortmund, Germany; 3. Medical Proteom-Center, Ruhr-University Bochum, D-44780 Bochum, Germany Correspondence to: Francisco Castaneda, MD, Laboratory for Molecular Pathobiochemistry and Clinical Research, Max Planck Institute for Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany; Tel. 49-231-9742-6490, Fax. 49-231-133-2699, E-mail: francisco.castaneda@mpi-dortmund.mpg.de Received: 2006.11.26; Accepted: 2006.12.15; Published: 2006.12.21 We previously found that ethanol at millimolar level (1 mM) activates the expression of transcription factors with subsequent regulation of apoptotic genes in human hepatocellular carcinoma (HCC) HepG2 cells. However, the role of ethanol on the expression of genes implicated in transcriptional and translational processes remains unknown. Therefore, the aim of this study was to characterize the effect of low concentration of ethanol on gene expression profiling in HepG2 cells using cDNA microarrays with especial interest in genes with transcriptional and translational function. The gene expression pattern observed in the ethanol-treated HepG2 cells revealed a relatively similar pattern to that found in the untreated control cells. The pairwise comparison analysis demonstrated four significantly up-regulated (COBRA1, ITGB4, STAU2, and HMGN3) genes and one down-regulated (ANK3) gene. All these genes exert their function on transcriptional and translational processes and until now none of these genes have been associated with ethanol. This functional genomic analysis demonstrates the reported interaction between ethanol and ethanol-regulated genes. Moreover, it confirms the relationship between ethanol-regulated genes and various signaling pathways associated with ethanol-induced apoptosis. The data presented in this study represents an important contribution toward the understanding of the molecular mechanisms of ethanol at low concentration in HepG2 cells, a HCC-derived cell line. Key words: human hepatocellular carcinoma cells, HepG2, ethanol, gene expression, transcriptional and translational processes 1. Introduction Studies using the human hepatocellular carcinoma (HCC) cell line HepG2 have demonstrated a specific gene expression pattern induced by ethanol different from that observed in normal livers and in livers with alcoholic liver disease [1, 2]. In vivo studies using animal models, including rats [3], mice [4], and baboons [5] as well as human liver samples obtained from patients with advanced alcoholic liver disease [5], revealed changes in the expression of genes coding for transcription factors, signaling molecules, stress response and Eukaryotic Transcription Eukaryotic Transcription Bởi: OpenStaxCollege Prokaryotes and eukaryotes perform fundamentally the same process of transcription, with a few key differences The most important difference between prokaryotes and eukaryotes is the latter’s membrane-bound nucleus and organelles With the genes bound in a nucleus, the eukaryotic cell must be able to transport its mRNA to the cytoplasm and must protect its mRNA from degrading before it is translated Eukaryotes also employ three different polymerases that each transcribe a different subset of genes Eukaryotic mRNAs are usually monogenic, meaning that they specify a single protein Initiation of Transcription in Eukaryotes Unlike the prokaryotic polymerase that can bind to a DNA template on its own, eukaryotes require several other proteins, called transcription factors, to first bind to the promoter region and then help recruit the appropriate polymerase The Three Eukaryotic RNA Polymerases The features of eukaryotic mRNA synthesis are markedly more complex those of prokaryotes Instead of a single polymerase comprising five subunits, the eukaryotes have three polymerases that are each made up of 10 subunits or more Each eukaryotic polymerase also requires a distinct set of transcription factors to bring it to the DNA template RNA polymerase I is located in the nucleolus, a specialized nuclear substructure in which ribosomal RNA (rRNA) is transcribed, processed, and assembled into ribosomes ([link]) The rRNA molecules are considered structural RNAs because they have a cellular role but are not translated into protein The rRNAs are components of the ribosome and are essential to the process of translation RNA polymerase I synthesizes all of the rRNAs except for the 5S rRNA molecule The “S” designation applies to “Svedberg” units, a nonadditive value that characterizes the speed at which a particle sediments during centrifugation 1/7 Eukaryotic Transcription Locations, Products, and Sensitivities of the Three Eukaryotic RNA Polymerases αAmanitin Sensitivity RNA Polymerase Cellular Product of Compartment Transcription I Nucleolus All rRNAs except 5S rRNA Nucleus All protein-coding Extremely nuclear presensitive mRNAs Nucleus 5S rRNA, tRNAs, and small nuclear RNAs II III Insensitive Moderately sensitive RNA polymerase II is located in the nucleus and synthesizes all protein-coding nuclear pre-mRNAs Eukaryotic pre-mRNAs undergo extensive processing after transcription but before translation For clarity, this module’s discussion of transcription and translation in eukaryotes will use the term “mRNAs” to describe only the mature, processed molecules that are ready to be translated RNA polymerase II is responsible for transcribing the overwhelming majority of eukaryotic genes RNA polymerase III is also located in the nucleus This polymerase transcribes a variety of structural RNAs that includes the 5S pre-rRNA, transfer pre-RNAs (pre-tRNAs), and small nuclear pre-RNAs The tRNAs have a critical role in translation; they serve as the adaptor molecules between the mRNA template and the growing polypeptide chain Small nuclear RNAs have a variety of functions, including “splicing” pre-mRNAs and regulating transcription factors A scientist characterizing a new gene can determine which polymerase transcribes it by testing whether the gene is expressed in the presence of a particular mushroom poison, α-amanitin ([link]) Interestingly, α-amanitin produced by Amanita phalloides, the Death Cap mushroom, affects the three polymerases very differently RNA polymerase I is completely insensitive to α-amanitin, meaning that the polymerase can transcribe DNA in vitro in the presence of this poison In contrast, RNA polymerase II is extremely sensitive to α-amanitin, and RNA polymerase III is moderately sensitive Knowing the transcribing polymerase can clue a researcher into the general function of the gene being studied Because RNA polymerase II transcribes the vast majority of genes, we will focus on this polymerase in our subsequent discussions about eukaryotic transcription factors and promoters 2/7 Eukaryotic Transcription Structure of an RNA Polymerase II Promoter Eukaryotic promoters are much larger and more complex than prokaryotic promoters, but both have a TATA box For example, in the mouse thymidine kinase gene, the TATA box is located at approximately -30 relative to the initiation (+1) site ([link]) For this gene, the exact TATA box sequence is TATAAAA, as read in the 5' to 3' direction on the nontemplate strand This sequence is not identical to the E coli TATA box, but it conserves the A–T rich element The thermostability of A–T bonds is low and this helps the DNA template to locally unwind in preparation for transcription A generalized promoter of a gene transcribed by RNA polymerase II is shown Transcription factors recognize the promoter RNA polymerase II then binds and forms the transcription initiation complex Art Connection 3/7 Eukaryotic ...A designed curved DNA segment that is a remarkable activator of eukaryotic transcription Noriyuki Sumida 1 , Jun-ichi Nishikawa 1 , Haruka Kishi 1 , Miho Amano 1 , Takayo Furuya 1 , Haruyuki Sonobe 1 and Takashi Ohyama 2,3 1 Department of Biology, Faculty of Science and Engineering, Konan University, Kobe, Japan 2 Department of Biology, School of Education, Waseda University, Tokyo, Japan 3 Graduate School of Science and Engineering, Waseda University, Tokyo, Japan DNA is packaged into chromatin in eukaryotes, thereby maintaining genes in an inactive state by restricting access to the general transcription machinery. Proteins that turn on or activate gene transcription are called activators, and these proteins recruit the chromatin remodeling complex to facilitate transcription [1–3]. Activators can bind to a target element in a regulatory promoter or enhancer [4], even when the target is adjacent to or actually within a nucleosome [5–9]. The regulatory promoter is typically located immediately upstream of the core promoter, which is positioned immediately adjacent to and upstream of the gene [4]. Regulatory promoters often include intrinsically curved DNA structures and poly(dAÆdT) sequences [10,11]. Recent studies have shown that such struc- tures are used to organize local chromatin structure to allow activator-binding sites to be accessible [11]. This suggests that engineering of chromatin structure for gene expression may be possible using these promoter structures or artificial mimics. This new technology, which might be referred to as ‘chromatin engineering’, would also permit stable expression of transgenes, which is of importance in many areas of the biological sciences. Moreover, such technology could lead to the development of useful nonviral vectors for gene ther- apy. Therefore, the goal of the current study was to construct artificial bent DNA segments that can stably express transgenes in the genome of living cells, as a first step in chromatin engineering. We have reported that a 36 bp left-handed curved DNA segment, which we refer to as T4 in the present study (T indicates a dTÆdA tract and the numeral indi- cates the number of tracts), activates the herpes sim- plex virus thymidine kinase (HSV tk) promoter in a Keywords chromatin; chromatin engineering; curved DNA; supercoil; transcription activator Correspondence T. Ohyama, Department of Biology, School of Education, Waseda University, 1-6-1 Nishi-Waseda, Shinjuku-ku, Tokyo 169-8050, Japan Fax: +81 3 3207 9694 Tel: +81 3 5286 1520 E-mail: ohyama@waseda.jp (Received 11 September 2006, revised 19 October 2006, accepted 25 October 2006) doi:10.1111/j.1742-4658.2006.05557.x To identify artificial DNA segments that can stably express transgenes in the genome of host cells, we built a series of curved DNA segments that mimic a left-handed superhelical structure. Curved DNA segments of 288 bp (T32) and 180 bp (T20) were able to activate transcription from the herpes simplex virus thymidine kinase (tk) promoter by approximately 150-fold and 70-fold, respectively, compared to a control in a transient transfection assay in COS-7 cells. The T20 segment was also able to activate transcription from the human adenovirus type 2 E1A promoter with an 18-fold increase in the same assay system, and also activated transcription from the tk promoter on episomes in COS-7 cells. We also established five HeLa cell lines with genomes containing T20 upstream of the transgene promoter and control cell lines with T20 deleted from the transgene locus. e 2010 Keystone Symposium focusing on mechanisms of eukaryotic transcriptional regulation featured a strong emphasis on genomic approaches. Many presentations included data that coupled chromatin immuno precipi- tation (ChIP) assays to deep sequencing (ChIP-seq) or tiling arrays (ChIP-chip) in order to map the locations of transcription factors and RNA polymerase II (Pol II) across the genomes of organisms from yeast to humans. In addition, biochemical techniques such as perman ga- nate footprinting, nuclear run-on, and nuclease digestion were applied to cellular chromatin and coupled to deep sequencing. Here, we group the presentations that focused primarily on genomics into three general cate- gories: promoter-proximal paused polymerases, chromatin and nucleosomes, and networks of transcriptional regulation. Promoter-proximal paused polymerases Historically, regulation of transcription was thought to occur primarily at the point of recruiting Pol II and its accessory factors to the promoters of genes. Now a battery of genomic studies is revealing that transcrip- tional regulation occurs at a post-initiation step at thou- sands of genes in both Drosophila and mammals. e signature of such genes is the presence of a promoter- proximal paused Pol II molecule - one that has initiated transcription and is poised for an activation signal in order to continue transcribing. Rick Young (Whitehead Institute and Massachusetts Institute of Technology, Cambridge, USA) in his keynote address provided a mechanism for how activation of paused polymerases can occur. In human embryonic stem cells the trans- cription factor c-Myc functions as a ‘pause release’ factor at approximately one-third of genes. ChIP-seq data have shown that c-Myc associates exclusively with transcribed genes near their start sites, a signature unique among stem cell transcription factors. Moreover, chemical inhibi tion of c-Myc, or its knockdown, decreased the elongating form of Pol II (phosphorylated on serine 2) but not the initiated form of Pol II (phosphorylated on serine 5). Karen Adelman (National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, USA) described her research showing that Pol II pausing followed by regulated release does not function solely as a transcriptional on/off switch. ChIP- chip studies showed that, in Drosophila cells, the pause- inducing factors negative elongation factor (NELF) and DRB sensitivity inducing factor (DSIF) occupy genes that are actively transcribed (genes with uniform Pol II distribution and with Ser2-phosphorylated Pol II). us, NELF is present at active genes and seems to be fine- tuning transcription. Computational analysis of NELF- dependent genes revealed an enrichment of GAGA sites, initiator (Inr), and TATA sequences, as well as a down- stream motif centered at the +30 position that could function to control pausing. In a unique application of deep sequencing technology, Dave Gilmour (Pennsyl- vania State University, University Park, USA) presented data that revealed regions of melted DNA associated with a paused polymerase. In vivo permanganate footprinting, which detects single-stranded thymines in the DNA comprising a transcription bubble, was coupled to Pol II ChIP-seq to reveal polymerases paused at specific positions across the Drosophila genome. At least 10% of genes showed permanganate reactivity centered in the +20 to +60 region, which correlates nicely with Gilmour’s in vitro biochemical data. Of these genes, 80% showed NELF occupancy and 50% had GAGA factor, consistent with these two factors controlling Pol II pausing. e pioneer of paused polymerases, John Lis (Cornell University, Ithaca, USA), described a powerful genomic technique coupling nuclear run-on assays to deep sequencing (GRO-seq) to map the location, density, and Abstract A report of the Keystone Symposium on Dynamics of Eukaryotic Genome Biology 2006, 7:323 comment reviews reports deposited research interactions information refereed research Meeting report An all-round view of eukaryotic transcription Tae Hoon Kim* and Bing Ren* † Addresses: *Ludwig Institute for Cancer Research, and † Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0653, USA. Correspondence: Bing Ren. Email: biren@ucsd.edu Published: 28 July 2006 Genome Biology 2006, 7:323 (doi:10.1186/gb-2006-7-7-323) The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2006/7/7/323 © 2006 BioMed Central Ltd A report of the Keystone Symposium ‘Regulation of Eukaryotic Transcription: From Chromatin to mRNA’, Taos, USA, 21-26 April 2006. Transcription of protein-coding genes in eukaryotes involves a complicated yet highly coordinated series of events involv- ing chromatin, chromatin modifiers, the transcriptional machineries and transcriptional regulators. A recent Key- stone Symposium on the regulation of eukaryotic transcrip- tion covered the topic from a variety of perspectives, both structural and biochemical. This report highlights some of the findings and new approaches reported at the meeting. Structural views of the transcriptional complex One key to understanding the mechanism of transcriptional initiation is an atomic-level view of the RNA polymerase pre- initiation complex (PIC). In his keynote address, Roger Kornberg (Stanford University, Palo Alto, USA) described new structural studies of a PIC containing the 12-subunit yeast RNA polymerase II (PolII) and general transcription factors bound to promoter DNA. From this we can see that TATA binding protein (TBP) configures DNA to the PolII surface; transcription factor II B (TFIIB) directs the DNA to the PolII active site and stabilizes the transcription complex; TFIIE recognizes the closed PolII complex and recruits the helicase TFIIH, while TFIIF captures the template strand DNA when the DNA duplex melts to form the transcriptional bubble. Finally, TFIIH introduces negative supercoiling of the promoter DNA, enabling the polymerase to move away from the promoter. Patrick Shultz (Institut de Génétique et de Biologie Molécu- laire et Cellulaire, Illkirch, France) has used cryo-electron microscopy to view yeast TFIID bound to DNA, revealing that DNA wraps around TFIID and threads through chan- nels formed between three structural modules. Eva Nogales (University of California, Berkeley, USA) has applied a new analytical approach to the cryo-electron microscopic struc- ture of human TFIID - three dimensional variance and con- formational flexibility analysis - and has characterized the structure in both closed and open forms. Transcription factor interactions The rate-limiting step to transcriptional initiation by PolII is promoter clearance. This is achieved when transcription proceeds independently of TFIIH, a short length of hybrid RNA-DNA has formed, and the initial transcription bubble collapses. Donald Luse (Cleveland Clinic Foundation, Cleve- land, USA) has found that stability of PolII on the promoter is minimal just before bubble collapse. He observed that the transcription bubble must be 17 nucleotides or longer, and the RNA transcript longer than six nucleotides, for the bubble to collapse, and that TFIIB must be phosphorylated; it is then displaced from the channel on PolII. Jim Kadonaga (University of California, San Diego, USA) presented a functional analysis of promoter sequence motifs from Drosophila and humans that are required for accurate transcription initiation, which revealed a network of interac- tion among these elements. MTE (motif 10), for example, can compensate for the loss of the downstream promoter element (DPE) and TATA by increasing the promoter’s affin- ity for TFIID. Kadonaga has constructed a ‘super’ core pro- moter containing TATA, the initiator motif VIETNAM NATIONAL UNIVERSITY, HANOI COLLEGE OF TECHNOLOGY Nguyen Trung Thong MEDSOFT, DECIPHERING PRINCIPLES OF TRANSCRIPTION REGULATION IN EUKARYOTIC GENOMES MASTER THESIS Hanoi - 2008 VIETNAM NATIONAL UNIVERSITY, HANOI COLLEGE OF TECHNOLOGY Nguyen Trung Thong MEDSOFT, DECIPHERING PRINCIPLES OF TRANSCRIPTION REGULATION IN EUKARYOTIC GENOMES Major: Information Technology Speciality: Computer science Code: 1.01.10 MASTER THESIS Advisor: Assoc. Prof. Hoang Xuan Huan Hanoi - 2008 4 Contents Abstract 1 Declaration 2 Acknowledgment 3 List of Figures 5 Glossary and abbreviations 6 Chapter 1 Introduction 7 1.1 Motivation 7 1.2 Thesis works and structure 9 Chapter 2 Transcription regulation in eukaryotic genomes 10 2.1 Introduction 10 2.1.1 Gene activation 10 2.1.2 Gene deactivation 12 2.2 Core promoter and basal transcription machinery 13 2.2.1 Structure of core promoter 14 2.2.2 Basal transcription machinery 16 2.3 Regulatory sequences 17 2.3.1 Enhancers and regulatory promoters 18 2.3.2 Activators 18 2.3.3 Repressors and corepressors 20 Chapter 3 Methods to derive principles of transcription regulation 21 3.1 Principles of transcription regulation 21 3.2 Typical methods to derive principles of transcription regulation 22 3.2.1 Bayesian network based method 22 3.2.2 Motif Expression Decomposition method 24 3.2.3 A comparison between two methods 26 Chapter 4 An application of MED method 30 4.1 MEDSoft workflow 31 4.2 Properties of MEDSoft 34 4.3 Experimental results 34 Chapter 5 Conclusions and Future work 40 Bibliography 41 Appendix 46 5 List of Figures Figure 1.1 Central dogma 7 Figure 2.1 Gene activation model 11 Figure 2.2 Sequence elements of core promoter 14 Figure 3.1 Gene regulatory network 22 Figure 3.2 Sequence elements that determine the regulation of a set of genes involved in transcription 23 Figure 3.3 The Motif-Expression Decomposition Formalism (MED) 25 Figure 3.4 An illustration of the concept of the gene ensemble 26 Figure 3.5 Verification model of regulatory principles 27 Figure 3.6 The distribution of correlation coefficients 28 Figure 3.7 RRPE and PAC relationship case study 29 Figure 4.1 MEDSoft layout 30 Figure 4.2 MEDSoft workflow 31 Figure 4.3 Genes and motifs query 32 Figure 4.4 Single motif analyzing 33 Figure 4.5 Pair of motifs analyzing 33 Figure 4.1 Transcriptional regulatory principle of RPN4 motif (short-range) 36 Figure 4.2 Transcriptional regulatory principle of MERE4 motif (short-range) 36 Figure 4.3 Transcriptional regulatory principle of GCR1 motif (middle-range) 36 Figure 4.4 Transcriptional regulatory principle of HAP234 motif (middle-range) 37 Figure 4.5 Transcriptional regulatory principle of BAS1 motif (long-range) 37 Figure 4.6 Transcriptional regulatory principle of GAL motif (long-range) 37 Figure 4.7 Transcriptional regulatory principle of PROTEOL1 motif (orientation-dependence) 38 Figure 4.8 Transcriptional regulatory principle of STRE’ motif (orientation- dependence) 38 Figure 4.9 Transcriptional regulatory principle of MIG1 motif (super-long- range) 38 Figure 4.10 Transcriptional regulatory principle of MERE17 motif (super-long- range) 39 Figure 4.11 Transcriptional regulatory principle of RPE11 motif (spread-out) . 39 6 Glossary and abbreviations Activator: protein product of a regulatory gene that induces expression of a target gene(s) usually by binding to the activation sequence of that gene or by interaction with transcription factors. Basal transcription: transcription in in vitro systems consisting of RNA polymerase, the basal transcription factors and naked DNA template; also used to describe in vivo transcription observed in the absence of known activators. Chromatin: the packaged eukaryotic chromosome in which the DNA is highly ... in our subsequent discussions about eukaryotic transcription factors and promoters 2/7 Eukaryotic Transcription Structure of an RNA Polymerase II Promoter Eukaryotic promoters are much larger.. .Eukaryotic Transcription Locations, Products, and Sensitivities of the Three Eukaryotic RNA Polymerases αAmanitin Sensitivity RNA Polymerase Cellular Product of Compartment Transcription. .. complex Art Connection 3/7 Eukaryotic Transcription Eukaryotic mRNA contains introns that must be spliced out A 5' cap and 3' poly-A tail are also added A scientist splices a eukaryotic promoter in