Eukaryotic Epigenetic 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 Epigenetic Gene Regulation Eukaryotic Epigenetic Gene Regulation Bởi: OpenStaxCollege Eukaryotic gene expression is more complex than prokaryotic gene expression because the processes of transcription and translation are physically separated Unlike prokaryotic cells, eukaryotic cells can regulate gene expression at many different levels Eukaryotic gene expression begins with control of access to the DNA This form of regulation, called epigenetic regulation, occurs even before transcription is initiated Epigenetic Control: Regulating Access to Genes within the Chromosome The human genome encodes over 20,000 genes; each of the 23 pairs of human chromosomes encodes thousands of genes The DNA in the nucleus is precisely wound, folded, and compacted into chromosomes so that it will fit into the nucleus It is also organized so that specific segments can be accessed as needed by a specific cell type The first level of organization, or packing, is the winding of DNA strands around histone proteins Histones package and order DNA into structural units called nucleosome complexes, which can control the access of proteins to the DNA regions ([link]a) Under the electron microscope, this winding of DNA around histone proteins to form nucleosomes looks like small beads on a string ([link]b) These beads (histone proteins) can move along the string (DNA) and change the structure of the molecule 1/5 Eukaryotic Epigenetic Gene Regulation DNA is folded around histone proteins to create (a) nucleosome complexes These nucleosomes control the access of proteins to the underlying DNA When viewed through an electron microscope (b), the nucleosomes look like beads on a string (credit “micrograph”: modification of work by Chris Woodcock) If DNA encoding a specific gene is to be transcribed into RNA, the nucleosomes surrounding that region of DNA can slide down the DNA to open that specific chromosomal region and allow for the transcriptional machinery (RNA polymerase) to initiate transcription ([link]) Nucleosomes can move to open the chromosome structure to expose a segment of DNA, but so in a very controlled manner Art Connection Nucleosomes can slide along DNA When nucleosomes are spaced closely together (top), transcription factors cannot bind and gene expression is turned off When the nucleosomes are spaced far apart (bottom), the DNA is exposed Transcription factors can bind, allowing gene expression to occur Modifications to the histones and DNA affect nucleosome spacing In females, one of the two X chromosomes is inactivated during embryonic development because of epigenetic changes to the chromatin What impact you think these changes would have on nucleosome packing? How the histone proteins move is dependent on signals found on both the histone proteins and on the DNA These signals are tags added to histone proteins and DNA that tell the histones if a chromosomal region should be open or closed ([link] depicts modifications to histone proteins and DNA) These tags are not permanent, but may be added or removed as needed They are chemical modifications (phosphate, methyl, 2/5 Eukaryotic Epigenetic Gene Regulation or acetyl groups) that are attached to specific amino acids in the protein or to the nucleotides of the DNA The tags not alter the DNA base sequence, but they alter how tightly wound the DNA is around the histone proteins DNA is a negatively charged molecule; therefore, changes in the charge of the histone will change how tightly wound the DNA molecule will be When unmodified, the histone proteins have a large positive charge; by adding chemical modifications like acetyl groups, the charge becomes less positive The DNA molecule itself can also be modified This occurs within very specific regions called CpG islands These are stretches with a high frequency of cytosine and guanine dinucleotide DNA pairs (CG) found in the promoter regions of genes When this configuration exists, the cytosine member of the pair can be methylated (a methyl group is added) This modification changes how the DNA interacts with proteins, including the histone proteins that control access to the region Highly methylated (hypermethylated) DNA regions with deacetylated histones are tightly coiled and transcriptionally inactive Histone proteins and DNA nucleotides can be modified chemically Modifications affect nucleosome spacing and gene expression (credit: modification of work by NIH) This type of gene regulation is called epigenetic regulation Epigenetic means “around genetics.” The changes that occur to the histone proteins and DNA not alter the nucleotide sequence and are not permanent Instead, these changes are temporary (although they often persist through multiple rounds of cell division) and alter the chromosomal structure (open or closed) as needed A gene can be turned on or off depending upon the location and modifications to the histone proteins and DNA If a gene is to be transcribed, the histone proteins and DNA are ...Characterization of the Drosophila Methoprene -tolerant gene product Juvenile hormone binding and ligand-dependent gene regulation Ken Miura, Masahito Oda, Sumiko Makita and Yasuo Chinzei Department of Medical Zoology, School of Medicine, Mie University, Tsu City, Japan Insect development and reproduction are regulated by two classes of lipid-soluble hormones, the ecdysteroids and juvenile hormones (JHs). The ecdysteroids activate target genes through a heterodimeric receptor complex composing the ecdysone receptor and ultraspiracle (USP) proteins, both of which are members of the nuc- lear steroid ⁄ thyroid ⁄ retinoid receptor superfamily [1]. During insect development, ecdysteroids induce molting while JH determines the nature of each molt by modu- lating the ecdysteroid-induced gene expression cascade [2–4]. In addition, in adult insects, JH has a wide variety of actions related to reproduction, including oogenesis, migratory behaviour and diapause [2,5,6]. The mode of molecular action of JH, however, is still obscure [7]. JHs are a family of esterified sesquiterpe- noids, whose lipid-soluble nature has suggested action directly on the genome through nuclear receptors such as ecdysteroids and the vertebrate steroid ⁄ thyroid ⁄ reti- noid hormones [5,8] although actions of JH through the cell membrane are also documented [9,10]. Many attempts have been made to identify nuclear JH receptors. Jones and Sharp [11] showed that JH III binds to the Drosophila USP protein, which is a homo- logue of the vertebrate retinoid X receptor, promoting Keywords juvenile hormone; juvenile hormone receptor; Methoprene-tolerant; Drosophila; transcription factor Correspondence K. Miura, Department of Medical Zoology, School of Medicine, Mie University, Edobashi 2-174, Tsu514-8507, Japan Fax: +81 59 231 5215 Tel: +81 59 231 5013 E-mail: k-miura@doc.medic.mie-u.ac.jp (Received 27 October 2004, revised 20 December 2004, accepted 4 January 2005) doi:10.1111/j.1742-4658.2005.04552.x Juvenile hormones (JHs) of insects are sesquiterpenoids that regulate a great diversity of processes in development and reproduction. As yet the molecular modes of action of JH are poorly understood. The Methoprene- tolerant (Met) gene of Drosophila melanogaster has been found to be responsible for resistance to a JH analogue (JHA) insecticide, methoprene. Previous studies on Met have implicated its involvement in JH signaling, although direct evidence is lacking. We have now examined the product of Met (MET) in terms of its binding to JH and ligand-dependent gene regu- lation. In vitro synthesized MET directly bound to JH III with high affinity (K d ¼ 5.3 ± 1.5 nm, mean ± SD), consistent with the physiological JH concentration. In transient transfection assays using Drosophila S2 cells the yeast GAL4-DNA binding domain fused to MET exerted JH- or JHA- dependent activation of a reporter gene. Activation of the reporter gene was highly JH- or JHA-specific with the order of effectiveness: JH III  JH II > JH I > methoprene; compounds which are only structur- ally related to JH or JHA did not induce any activation. Localization of MET in the S2 cells was nuclear irrespective of the presence or absence of JH. These results suggest that MET may function as a JH-dependent tran- scription factor. Abbreviations Ahr, aryl hydrocarbon receptor; Arnt, Ahr nuclear translocator; bHLH, basic helix-loop-helix; DBD, DNA binding domain; DCC, dextran-coated charcoal; EGFP, enhanced green REVIEW ARTICLE Functional interplay between viral and cellular SR proteins in control of post-transcriptional gene regulation Ming-Chih Lai 1, *, Tsui-Yi Peng 1,2, * and Woan-Yuh Tarn 1 1 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 2 Institute of Molecular Medicine, National Tsing Hua University, Hsin-Chu, Taiwan Introduction Arginine ⁄ serine (RS) dipeptide repeats are present in a number of cellular proteins, termed SR proteins, that primarily participate in nuclear precursor (pre)-mRNA splicing [1–3]. RS domain variants, such as serine and arginine-rich motifs or arginine–aspartate or arginine– glutamate dipeptide-rich domains, are also found in many nuclear proteins. In addition to the RS domains, SR splicing factors often contain one or more RNA recognition motifs. SR proteins function in both constitutive and regulated splicing via binding to cis-elements of pre-mRNA or interaction with other splicing factors. The RS domain interacts with both proteins and RNAs [1–3]. In particular, intermolecular interactions between SR proteins, which are important for spliceosome assembly and splice site determination during pre-mRNA splicing, are mediated by their RS domains [3]. The RS domain also acts as a nuclear localization signal and targets SR proteins to nuclear speckled domains, where splicing factors are concen- trated, for storage [1]. An important biochemical property of the RS domain is its differential phosphorylation at multiple serine and threonine residues. The RS domain is primarily phos- phorylated by SR protein-specific kinases (SRPKs), and Keywords Alternative splicing; kinases; phosphatases; phosphorylation; post-transcriptional control; pre-mRNA splicing; RS domain; SR proteins; viral problems; virus Correspondence W Y. Tarn, Institute of Biomedical Sciences, Academia Sinica, 128 Academy Road, Section 2, Nankang, Taipei 11529, Taiwan Fax: +886 2 2782 9142 Tel: +886 2 2652 3052 E-mail: wtarn@ibms.sinica.edu.tw *These authors contributed equally to this work (Received 3 November 2008, revised 14 December 2008, accepted 9 January 2009) doi:10.1111/j.1742-4658.2009.06894.x Viruses take advantage of cellular machineries to facilitate their gene expression in the host. SR proteins, a superfamily of cellular precursor mRNA splicing factors, contain a domain consisting of repetitive argi- nine ⁄ serine dipeptides, termed the RS domain. The authentic RS domain or variants can also be found in some virus-encoded proteins. Viral pro- teins may act through their own RS domain or through interaction with cellular SR proteins to facilitate viral gene expression. Numerous lines of evidence indicate that cellular SR proteins are important for regulation of viral RNA splicing and participate in other steps of post-transcriptional viral gene expression control. Moreover, viral infection may alter the expression levels or modify the phosphorylation status of cellular SR proteins and thus perturb cellular precursor mRNA splicing. We review our current understanding of the interplay between virus and host in post-transcriptional regulation of gene expression via RS domain-containing proteins. Abbreviations CTE, constitutive transport element; E4, early region 4; EV, epidermodysplasia verruciformis; HBV, hepatitis B virus; HCV, hepatitis C virus; hnRNP, heterogeneous nuclear ribonucleoprotein; HPV, human papillomavirus; HSV, herpes simplex virus; IRES, internal ribosome entry site; N, nucleocapsid; PP, protein phosphatase; REVIEW ARTICLE Gene regulation by tetracyclines Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes Christian Berens and Wolfgang Hillen Lehrstuhl fu ¨ r Mikrobiologie, Institut fu ¨ r Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander Universita ¨ t Erlangen-Nu ¨ rnberg; Germany The Tet repressor protein (TetR) regulates transcription of a family of tetracycline (tc) resistance determinants in Gram-negative bacteria. The resistance protein TetA, a membrane-spanning H + -[tcÆM] + antiporter, must be sen- sitively regulated because its expression is harmful in the absence of tc, yet it has to be expressed before the drugs’ concentration reaches cytoplasmic levels inhibitory for protein synthesis. Consequently, TetR shows highly speci- fic tetO binding to reduce basal expression and high affinity to tc to ensure sensitive induction. Tc can cross biological membranes by diffusion enabling this inducer to penetrate the majority of cells. These regulatory and pharmacological properties are the basis for application of TetR to selec- tively control the expression of single genes in lower and higher eukaryotes. TetR can be used for that purpose in some organisms without further modifications. In mam- mals and in a large variety of other organisms, however, eukaryotic transcriptional activator or repressor domains are fused to TetR to turn it into an efficient regulator. Mechanistic understanding and the ability to engineer and screen for mutants with specific properties allow tailoring of the DNA recognition specificity, the response to inducer tc and the dimerization specificity of TetR-based eukary- otic regulators. This review provides an overview of the TetR properties as they evolved in bacteria, the functional modifications necessary to transform it into a convenient, specific and efficient regulator for use in eukaryotes and how the interplay between structure ) function studies in bacteria and specific requirements of particular applica- tions in eukaryotes have made it a versatile and highly adaptable regulatory system. Keywords: antibiotic resistance; disease models; fusion pro- tein; inducible gene expression; ligand-binding specificity; mammalian cell lines; protein engineering; structure–activity relationship; Tet repressor; transgenic organism. Properties of bacterial Tet systems Efflux-mediated tetracycline resistance is always regulated in Gram-negative bacteria In Gram-negative bacteria, resistance to tetracyclines (tc) is mediated by the TetA protein, a proton-[tcÆMg] + anti- porter embedded in the cytoplasmic membrane [1,2]. Eleven tc resistance determinants (Tet classes A–E, G, H, J, Z, 30, and 33 [3–5]) share the organization of structural and regulatory genes (reviewed in [6]). In enteric bacteria, the efflux-encoding tetA genes are strictly regulated at the level of transcription by the tc-responsive Tet repressor (TetR). In the absence of inducer, TetR dimers bind to the operators tetO 1 and tetO 2 , shutting down transcription of its own gene, tetR, and of the resistance gene, tetA.Oncetchas entered the cell, it binds TetR with high affinity as a [tcÆMg] + complex [7]. This induces a conformational change in TetR [8] resulting in dissociation from tetO [9]. The following expression burst of TetA and TetR leads to a rapid reduction of the cytoplasmic tc concentration [10] which, in turn, shuts expression of both genes off again. Expression MINIREVIEW Collective behavior in gene regulation: Metabolic clocks and cross-talking Michele M. Bianchi Department of Cell and Developmental Biology, University of Rome ‘La Sapienza’, Italy By cosmic rule, as day yields night, so winter sum- mer, war peace, plenty famine. All things change… the harmonious structure of the world depends upon opposite tensions. (Heraclitus, 500 bc) In the modern age, life scientists subscribe to the ergo- dic cell hypothesis (Fig. 1): they use homogenized tissues or cultured cells, analyze extracts and draw conclusions about a hypothetical representative cell on the basis that all cells are ‘on average’ identical over (short) time and space scales [1]. In this representation (statistical mechanics, where it allowed a microscopic basis to be given to thermodynamics), the average of a process parameter for a single cell over time and the average over the statistical ensemble of individuals at a given time coincide. In the ergodic hypothesis, genes are generally divided into housekeeping genes, which are always expressed, and regulated genes, which are expressed or repressed under the effect of external signals. The external signal might have various origins: an environ- mental condition, a physiological signal from other regions of a multicellular organism, the result of a developmental programme, epigenetic control and so on. In any case, these external signals occur inciden- tally and ‘on average’ elicit the same response in all cells; this means that they may have different effects depending on the status of each cell but, given that the population is very large and a point in time displays the same distribution of states, the average result is the same irrespective of time. If we want to study the behavior of a single cell in a time-dependent manner, by analysing a representative population of individuals, we must artificially put all the cells into the same state by synchronization, in order to collapse the ensemble distribution into a single state. This collapse is usually unstable and, after a relatively short time, the cell pop- ulation reverts to the statistical distribution of states. Keywords circadian clock; cross-talk; cycles; ergodic system; message; metabolism; redox; synchronization; transcription dynamics; ultradian clock Correspondence M. M. Bianchi, Department of Cell and Developmental Biology, p.le Aldo Moro 5, 00185 Rome, Italy Fax: +39 064 991 2351 Tel: +39 064 991 2215 E-mail: michele.bianchi@uniroma1.it (Received 10 December 2007, accepted 30 January 2008) doi:10.1111/j.1742-4658.2008.06397.x Biological functions governed by the circadian clock are the evident result of the entrainment operated by the earth’s day and night cycle on living organisms. However, the circadian clock is not unique, and cells and organisms possess many other cyclic activities. These activities are difficult to observe if carried out by single cells and the cells are not coordinated but, if they can be detected, cell-to-cell cross-talk and synchronization among cells must exist. Some of these cycles are metabolic and cell syn- chronization is due to small molecules acting as metabolic messengers. We propose a short survey of cellular cycles, paying special attention to meta- bolic cycles and cellular cross-talking, particularly when the synchroniza- tion of metabolism or, more generally, cellular functions are concerned. Questions arising from the observation of phenomena based on cell com- munication and from basic cellular cycles are also proposed. Abbreviations ROS, reactive oxygen species; YGO, yeast glycolytic oscillation; YMC, yeast metabolic cycle. 2356 FEBS Journal 275 (2008) 2356–2363 ª 2008 The Author Journal compilation ª 2008 FEBS Clocks Looking closer at the cell or organism and ... that describes how epigenetic regulation controls gene expression Section Summary In eukaryotic cells, the first stage of gene expression control occurs at the epigenetic level Epigenetic mechanisms... affect nucleosome spacing and gene expression (credit: modification of work by NIH) This type of gene regulation is called epigenetic regulation Epigenetic means “around genetics.” The changes that... are modified surrounding the chromosomal region encoding that gene This opens the chromosomal region to 3/5 Eukaryotic Epigenetic Gene Regulation allow access for RNA polymerase and other proteins,