DOCSLIB.ORG

Analysis of the DNA-Binding and Activation Properties of the Human Transcription Factor AP-2

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Analysis of the DNA-Binding and Activation Properties of the Human Transcription Factor AP-2 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Analysis of the DNA-binding and activation properties of the human transcription factor AP-2 Trevor Williams ~ and Robert Tjian Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 USA The mammalian transcription factor AP-2 is a sequence-specific DNA-binding protein expressed in neural crest lineages and regulated by retinoic acid. Here we report a structure/function analysis of the DNA-binding and transcription activation properties of the AP-2 protein. DNA contact studies indicate that AP-2 binds as a dimer to a palindromic recognition sequence. Furthermore, cross-linking and immunoprecipitation data illustrate that AP-2 exists as a dimer even in the absence of DNA. Examination of cDNA mutants reveals that the sequences responsible for DNA binding are located in the carboxy-terminal half of the protein. In addition, a domain mediating dimerization forms an integral component of this DNA-binding structure. Expression of AP-2 in mammalian cells demonstrates that transcriptional activation requires an additional amino-terminal domain that contains an unusually high concentration of proline residues. This proline-rich activation domain also functions when attached to the heterologous DNA-binding region of the GAL4 protein. This study reveals that although AP-2 shares an underlying modular organization with other transcription factors, the regions of AP-2 involved in transcriptional activation and DNA binding/dimerization have novel sequence characteristics. [Key Words: Enhancer factor~ DNA binding~ dimerization; mammalian expression~ proline-rich activator~ palindromic binding site] Received November 15, 19901 revised version accepted January 22, 1991. Sequence-specific DNA-binding proteins form a vital post-translationally {Mitchell et al. 1987). The AP-2 pro- link between cis-regulatory DNA elements, present in tein binds to a GC-rich recognition sequence present in promoter and enhancer sequences, and the general tran- the cis-regulatory regions of several viral and cellular scription machinery. Modulation of the concentration genes, including the enhancers of SV40, HTLV-1, human and activity of these proteins provides a fundamental metallothionein IIa, and mouse major histocompatibil- mechanism for regulating gene expression. Alterations ity complex (MHC} H-2K b (Imagawa et al. 1987~ Mitchell in the pattern of expression of DNA-binding transcrip- et al. 1987; Nyborg and Dynan 1990}. In addition, AP-2 tion factors can lead to new programs of gene transcrip- can stimulate transcription in a binding site-dependent tion during development. The modification of the activ- manner both in vivo and in vitro {Mitchell et al. 1987~ ity of a pre-existing transcription factor represents one of Williams et al. 1988). Interestingly, the expression of AP- the fastest methods for a cell to alter its pattern of gene 2 mRNA and protein is stimulated by retinoic acid-in- expression in response to extracellular stimuli. Further- duced differentiation of human NT2 teratocarcinoma more, aberrant transcription factor expression can be as- cells {Williams et al. 1988~ Ltischer et al. 1989}. Further- sociated with oncogenesis. The delineation of the do- more, AP-2 is expressed in a cell-type-specific manner~ mains responsible for DNA-binding and transcriptional AP-2 is absent in the human hepatoma cell line HepG2 activation provides important clues toward understand- but present in human HeLa fibroblast cells (Williams et ing how these factors influence gene expression (Ptashne al. 1988}. More recent data also show that AP-2 mRNA 1988~ Mitchell and Tjian 1989). levels are regulated both temporally and spatially during The mammalian enhancer-binding protein AP-2 pre- mouse embryogenesis (Mitchell et al. 1991}. Intriguing- sents an attractive system to study the regulation of ly, the highest levels of expression appear to correspond transcription factor activity. This protein is controlled at to early neural crest cells and suggest that AP-2 plays a the level of its own gene expression (Williams et al. role in their subsequent differentiation and develop- 1988~ Lfischer et al. 1989) and may also be regulated ment. The activity of the AP-2 protein also appears to be 1Corresponding author. controlled post-translationally by both positive and neg- 670 GENES & DEVELOPMENT 5:670-682 © 1991 by Cold Spring Harbor Laboratory ISSN 0890-9369/91 $3.00 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Structure/function analysis of AP-2 ative regulatory mechanisms. Several studies suggest are summarized in Figure 1A. These studies reveal the that AP-2 is displaced from its recognition site by com- occurrence of a consensus sequence, 5'-GN4GGG-3', petition from adjacent DNA-binding proteins (Isra61 et present on one strand of both the SV40 and hMtIIa-bind- al. 1989; Mercurio and Karin 1989; Courtois et al. 1990). ing sites. Furthermore, a slightly degenerate copy of this The DNA-binding activity of AP-2 can also be blocked sequence also occurs on the two complementary strands. by the SV40 large T antigen oncogene product (Mitchell Thus, the AP-2-binding site appears to contain a dyad et al. 1987). In contrast, the ability of AP-2 to activate repeat. Methylation of the guanine present at the center gene expression is stimulated by the hepatitis B virus X of the dyad repeat does not affect AP-2 DNA binding. In protein (Seto et al. 1990). The AP-2 DNA recognition contrast, methylation of the flanking guanines present in sequence also seems to act as both a TPA- and cAMP- the 5'-GN4GGG-3' sequence reduces AP-2 binding to inducible element (Imagawa et al. 1987; Roesler et al. these sites. 1988). To determine the contribution of all the individual The examination of AP-2 activity has been facilitated bases within and around the binding sites we also per- by the recent cloning of a human cDNA encoding the formed missing contact probing assays. This procedure is entire AP-2 open reading frame (Williams et al. 1988). used to determine how the removal of a particular base Expression of the full-length clone in Escherichia coli from the recognition site affects DNA binding. The ex- generated a protein that was indistinguishable from the perimental protocol was similar to methylation interfer- endogenous HeLa factor in its ability to bind DNA and ence except that the starting DNA template was par- activate transcription. Here, we report the analysis of the tially depurinated or depyrimidinated. Representative re- DNA-binding and transcriptional activation properties sults obtained from this analysis are shown in Figure 1B, of the AP-2 protein using a series of cDNA mutants. This and the data are summarized schematically in Figure 1C. analysis reveals that AP-2 has a modular organization The data indicate that many bases within both the SV40 with separate domains involved in transcriptional acti- and hMtIIa sequences are needed to generate a functional vation and DNA binding/dimerization. The information AP-2 binding site. In addition, missing contact probing of obtained should enable us to dissect the mechanism of the SV40 AP-2-site illustrates that flanking DNA se- AP-2 action and understand how it may be regulated. quences may also be important in the generation of a functional binding site. Interestingly, however, the re- moval of symmetrically located cytosine bases within Results the binding site does not affect AP-2 DNA binding (SV40 base pairs 233 and 237; hMtIIa base pairs -177 and The AP-2 recognition sequence contains a dyad repeat -173). Similarly, the cytosine present at the center of The AP-2 protein must perform a minimum of two func- dyad symmetry is not important for protein/DNA con- tions to act as a sequence-specific DNA-binding tran- tact. Molecular modeling indicates that these three cy- scription factor. First, AP-2 must be able to recognize tosine bases can all be aligned on one face of the DNA and bind to a specific DNA sequence; second, the protein helix and suggests that AP-2 is not contacting this region must recruit the general transcription machinery to in- of the DNA (Fig. 1D). It is also interesting to note that crease the rate of initiation. To analyze the former pro- the missing contact probing and methylation interfer- cess we have examined which components of the AP-2 ence procedures measure different aspects of DNA bind- recognition sequence are important for specific protein/ ing, as AP-2 can bind to the recognition sequence when DNA contact. Previously, DNase I footprinting experi- the guanine at the center of dyad symmetry is methyl- ments have shown that AP-2 binds to a GC-rich se- ated but not when it is removed. Taken together, the quence that is present in a variety of promoter and en- results show that the SV40 and hMtIIa sites share several hancer elements associated with both viral and cellular common sequence features. Moreover, the data allow genes (Imagawa et al. 1987; Mitchell et al. 1987). How- the identification of a palindromic sequence, 5'-GCCN3- ever, DNase I footprinting does not generate information GGC-3', which appears to form the core recognition el- concerning the contribution of individual nucleotides ement for the AP-2-binding site. within the DNA-binding site. Therefore, to obtain a more detailed understanding of AP-2 DNA binding, we The carboxy-terminal region of the AP-2 protein performed a series of methylation interference assays us- mediates DNA binding ing the AP-2 sites present in the SV40 enhancer and the human metallothionein IIa distal basal level element The determination of which nucleotides in the recogni- (hMtIIa BLE). Methylation interference assesses the tion site are involved in AP-2 DNA binding represents steric inhibition of DNA binding caused by the presence one aspect in the analysis of protein-DNA interaction.
Load more

Recommended publications
  • Locating Mammalian Transcription Factor Binding Sites: a Survey of Computational and Experimental Techniques
    Downloaded from genome.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Review Locating mammalian transcription factor binding sites: A survey of computational and experimental techniques Laura Elnitski,1,4 Victor X. Jin,2 Peggy J. Farnham,2 and Steven J.M. Jones3 1Genomic Functional Analysis Section, National Human Genome Research Institute, National Institutes of Health, Rockville, Maryland 20878, USA; 2Genome and Biomedical Sciences Facility, University of California–Davis, Davis, California 95616-8645, USA; 3Genome Sciences Centre, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada V5Z-4E6 Fields such as genomics and systems biology are built on the synergism between computational and experimental techniques. This type of synergism is especially important in accomplishing goals like identifying all functional transcription factor binding sites in vertebrate genomes. Precise detection of these elements is a prerequisite to deciphering the complex regulatory networks that direct tissue specific and lineage specific patterns of gene expression. This review summarizes approaches for in silico, in vitro, and in vivo identification of transcription factor binding sites. A variety of techniques useful for localized- and high-throughput analyses are discussed here, with emphasis on aspects of data generation and verification. [Supplemental material is available online at www.genome.org.] One documented goal of the National Human Genome Research and other regulatory elements is mediated by DNA/protein in- Institute (NHGRI) is the identification of all functional noncod- teractions. Thus, one major step in the characterization of the ing elements in the human genome (ENCODE Project Consor- functional elements of the human genome is the identification tium 2004).
    [Show full text]
  • Specificity Landscapes Unmask Submaximal Binding Site Preferences of Transcription Factors
    Specificity landscapes unmask submaximal binding site preferences of transcription factors Devesh Bhimsariaa,b,1, José A. Rodríguez-Martíneza,2, Junkun Panc, Daniel Rostonc, Elif Nihal Korkmazc, Qiang Cuic,3, Parameswaran Ramanathanb, and Aseem Z. Ansaria,d,4 aDepartment of Biochemistry, University of Wisconsin–Madison, Madison, WI 53706; bDepartment of Electrical and Computer Engineering, University of Wisconsin–Madison, Madison, WI 53706; cDepartment of Chemistry, University of Wisconsin–Madison, Madison, WI 53706; and dThe Genome Center of Wisconsin, University of Wisconsin–Madison, Madison, WI 53706 Edited by Michael Levine, Princeton University, Princeton, NJ, and approved September 24, 2018 (received for review July 13, 2018) We have developed Differential Specificity and Energy Landscape Here, we report the development of Differential Specificity (DiSEL) analysis to comprehensively compare DNA–protein inter- and Energy Landscapes (DiSEL) to compare experimental actomes (DPIs) obtained by high-throughput experimental plat- platforms, computational methods, and interactomes of TFs, forms and cutting edge computational methods. While high-affinity especially those factors that bind identical consensus motifs. Our DNA binding sites are identified by most methods, DiSEL uncovered results reveal that (i) most high-throughput experimental plat- nuanced sequence preferences displayed by homologous transcription forms reliably identify high-affinity motifs but yield less reliable factors. Pairwise analysis of 726 DPIs uncovered homolog-specific dif- information on submaximal sites; (ii) with few exceptions, com- ferences at moderate- to low-affinity binding sites (submaximal sites). putational methods model DPIs with a focus on high-affinity DiSEL analysis of variants of 41 transcription factors revealed that sites; (iii) submaximal sites improve the annotation of biologi- many disease-causing mutations result in allele-specific changes in cally relevant binding sites across genomes; (iv) among members binding site preferences.
    [Show full text]
  • DNA Binding Site of the Growth Factor-Inducible Protein Zif268
    Proc. Natl. Acad. Sci. USA Vol. 86, pp. 8737-8741, November 1989 Biochemistry DNA binding site of the growth factor-inducible protein Zif268 ("zinc fingers"/transcription factor/cell growth) BARBARA CHRISTY AND DANIEL NATHANS* Howard Hughes Medical Institute and the Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205 Contributed by Daniel Nathans, August 31, 1989 ABSTRACT Zif268, a zinc finger protein whose mRNA is The recombinant plasmid was transfected into E. coli strain rapidly activated in cells exposed to growth factors or other BL21(DE3) and the transfected cells were grown as described signaling agents, is thought to play a role in regulating the (22) to an optical density of about 0.6 before induction of T7 genetic program induced by extracellular ligands. We report RNA polymerase with 0.4 mM isopropyl B-D-thiogalacto- that Zif268 has one of the characteristics of a transcriptional pyranoside (IPTG). Bacterial extracts used for DNA binding regulator, namely, sequence-specific binding to DNA. Zif268 were prepared as described (23). After solubilization of synthesized in Escherichia coli bound to two sites upstream of proteins with 4 M urea the preparation was dialyzed against the zif268 gene and to sites in the promoter regions of other 20 mM Tris, pH 7.7/50 mM KCI/10 mM MgCl2/i mM genes. The nucleotide sequences responsible for binding were EDTA/10 ,uM ZnSO4/20% (vol/vol) glycerol/1 mM dithio- defined by DNase I footprinting, by methylation interference threitol/0.2 mM phenylmethylsulfonyl fluoride/1 mM so- experiments, and by use of synthetic oligonucleotides.
    [Show full text]
  • Direct Inhibition of the DNA-Binding Activity of POU Transcription Factors
    Published online 25 April 2008 Nucleic Acids Research, 2008, Vol. 36, No. 10 3341–3353 doi:10.1093/nar/gkn208 Direct inhibition of the DNA-binding activity of POU transcription factors Pit-1 and Brn-3 by selective binding of a phenyl-furan-benzimidazole dication Paul Peixoto1,2, Yang Liu3, Sabine Depauw1,2, Marie-Paule Hildebrand1,2,4, David W. Boykin3, Christian Bailly1,2, W. David Wilson3 and Marie-He´ le` ne David-Cordonnier1,2,* Downloaded from https://academic.oup.com/nar/article/36/10/3341/2410535 by guest on 23 September 2021 1INSERM U-837, Team 4-‘Molecular and cellular targeting for cancer treatment’, Jean-Pierre Aubert Research Center, Institut de Recherches sur le Cancer de Lille, Place de Verdun, F-59045 Lille, 2IMPRT-IFR114, Lille, France, 3Department of Chemistry, Georgia State University, Atlanta, GA, USA and 4Institut de Recherches sur le Cancer de Lille - IRCL, Lille, France Received February 5, 2008; Revised and Accepted April 8, 2008 ABSTRACT control of transcription factors activity by those The development of small molecules to control gene compounds. expression could be the spearhead of future- targeted therapeutic approaches in multiple pathol- ogies. Among heterocyclic dications developed with INTRODUCTION this aim, a phenyl-furan-benzimidazole dication The aim of exerting precise control over the expression DB293 binds AT-rich sites as a monomer and level of specified genes using a small molecule drug is an ’ 5 -ATGA sequence as a stacked dimer, both in the objective with major consequences for many therapeutic minor groove. Here, we used a protein/DNA array applications including cancer, chronic inflammatory dis- approach to evaluate the ability of DB293 to specif- orders, neuro-degenerative or cardiovascular diseases ically inhibit transcription factors DNA-binding in a (1–4).
    [Show full text]
  • A Region of Phage Lambda Required for Antitermination of Transcription
    Cell, Vol. 31, 61-70, November 1982, Copyright 0 1982 by MIT Analysis of nutR; A Region of Phage Lambda Required for Antitermination of Transcription Eric R. Olson, Eric L. Flamm* and 1971; Friedman and Baron, 1974; Keppel et al., David I. Friedman 1974; Friedman et al., 1976, 1981; Greenblatt et al., Department of Microbiology and Immunology 1980). The sites include putative sites of pN action, The University of Michigan Medical School called nut (Salstrom and Szybalski, 1978; Rosenberg Ann Arbor, Michigan 48109 et al., 1978), as well as termination signals (Roberts, 1969; Rosenberg et al., 1978; Rosenberg and Court, 1979). Summary Consideration of the nature of the various factors involved in pN action formed the basis for the following The N gene product of coliphage lambda acts with model of pN action (discussed by Friedman and host factors (Nus) through sites (not) to render Gottesman, 1982). Transcription initiating at the early subsequent downstream transcription resistant to promoters PR and P, extends through the n&R and a variety of termination signals. These sites, not!? nutl sites, respectively (Figure 1). At these sites RNA and nutL, are downstream, respectively, from the polymerase is modified, rendering continuing tran- early promoters PR and PL. Thus a complicated set scription resistant to termination signals. The nature of molecular interactions are likely to occur at the of the promoter appears to play no role in pN action, nut sites. We have selected mutations in the nutR since placement of the n&R region downstream from region that reduce the effectiveness of pN in alter- the bacterial gal operon promoter results in termina- ing transcription initiating at the PR promoter.
    [Show full text]
  • A Framework for Automated Enrichment of Functionally Significant Inverted Repeats in Whole Genomes
    Missouri University of Science and Technology Scholars' Mine Computer Science Faculty Research & Creative Works Computer Science 01 Feb 2010 A Framework for Automated Enrichment of Functionally Significant Inverted Repeats in Whole Genomes Cyriac Kandoth Fikret Ercaļ Missouri University of Science and Technology, [email protected] Ronald L. Frank Missouri University of Science and Technology, [email protected] Follow this and additional works at: https://scholarsmine.mst.edu/comsci_facwork Part of the Biology Commons, and the Computer Sciences Commons Recommended Citation C. Kandoth et al., "A Framework for Automated Enrichment of Functionally Significant Inverted Repeats in Whole Genomes," BMC Bioinformatics, vol. 11, no. SUPPL. 6, BioMed Central Ltd., Feb 2010. The definitive version is available at https://doi.org/10.1186/1471-2105-11-S6-S20 This Article - Conference proceedings is brought to you for free and open access by Scholars' Mine. It has been accepted for inclusion in Computer Science Faculty Research & Creative Works by an authorized administrator of Scholars' Mine. This work is protected by U. S. Copyright Law. Unauthorized use including reproduction for redistribution requires the permission of the copyright holder. For more information, please contact [email protected]. Kandoth et al. BMC Bioinformatics 2010, 11(Suppl 6):S20 http://www.biomedcentral.com/1471-2105/11/S6/S20 PROCEEDINGS Open Access A framework for automated enrichment of functionally significant inverted repeats in whole genomes Cyriac Kandoth1*, Fikret Ercal1†, Ronald L Frank2† From Seventh Annual MCBIOS Conference. Bioinformatics: Systems, Biology, Informatics and Computation Jonesboro, AR, USA. 19-20 February 2010 Abstract Background: RNA transcripts from genomic sequences showing dyad symmetry typically adopt hairpin-like, cloverleaf, or similar structures that act as recognition sites for proteins.
    [Show full text]
  • Preferences in a Trait Decision Determined by Transcription Factor Variants
    Preferences in a trait decision determined by transcription factor variants Michael W. Dorritya,b, Josh T. Cuperusa,c, Jolie A. Carlislea, Stanley Fieldsa,c,d,1, and Christine Queitscha,1 aDepartment of Genome Sciences, University of Washington, Seattle, WA 98195; bDepartment of Biology, University of Washington, Seattle, WA 98195; cHoward Hughes Medical Institute, University of Washington, Seattle, WA 98195; and dDepartment of Medicine, University of Washington, Seattle, WA 98195 Contributed by Stanley Fields, June 27, 2018 (sent for review April 9, 2018; reviewed by Leah E. Cowen and Andrew W. Murray) Few mechanisms are known that explain how transcription factors mating, Ste12 can bind at pheromone-responsive genes as a can adjust phenotypic outputs to accommodate differing environ- homodimer or with the cofactors Mcm1 or Matα1 (10–13). The ments. In Saccharomyces cerevisiae, the decision to mate or invade consensus DNA-binding site of Ste12 is TGAAAC, known as the relies on environmental cues that converge on a shared transcription pheromone response element (PRE) (11). For invasion, Ste12 factor, Ste12. Specificity toward invasion occurs via Ste12 binding and its cofactor Tec1 are both required to activate genes that me- cooperatively with the cofactor Tec1. Here, we determine the range diate filamentation (8, 14, 15). Some of these genes contain a of phenotypic outputs (mating vs. invasion) of thousands of DNA- Ste12 binding site near a Tec1 consensus sequence of GAATGT, an binding domain variants in Ste12 to understand how preference for organization for heterodimeric binding known as a filamentation invasion may arise. We find that single amino acid changes in the response element (FRE).
    [Show full text]
  • DNA Sequence; Operator; Repressor; Regulatory Region; Dyad Symmetry; Plasmid Pbr322; Sl Nuclease Mapping)
    Gene, 23 (1983) 149-156 149 Elsevier Overlapping divergent promoters control expression of TnlO tetracycline resistance (DNA sequence; operator; repressor; regulatory region; dyad symmetry; plasmid pBR322; Sl nuclease mapping) Kevin P. Bertrand *, Kathleen Posh, Lewis V. Wray Jr. ** and William S. Reznikoff ** Department of Microbiology, University of California, Irvine, CA 92717 (U.S.A.) Tel. (714) 833- 6115, and ** Depart- ment of Biochemistry, University of Wisconsin, Madison, WI 53706 (U.S.A.) Tel. (608) 262 - 3608 (Received February 7th, 1983) (Accpeted February l&h, 1983) SUMMARY We have previously examined the genetic organization and regulation of the TnZO tetracycline-resistance determinant in Escherichia coli K-12. The structural genes for retA, the TnlO tetracycline-resistance function, and for tetR, the TnlO lef repressor, are transcribed in opposite directions from promoters in a regulatory region located between the two structural genes. Expression of both t&I and t&R is induced by tetracycline. Here we report the DNA sequence of the TnlO tet regulatory region. The locations of the tetA and tetR promoters within this region were defined by Sl nuclease mapping of the 5’ ends of in vivo tet RNA. The t&4 and tetR promoters overlap; the transcription start points are separated by 36 bp. We propose that two similar regions of dyad symmetry within the TnlO tet regulatory region are operator sites at which tet repressor binds to tet DNA, thereby in~biting transcription initiation at the ietA and tetR promoters. The TnlO ret regulatory region and the pBR322 tet regulatory region show significant DNA sequence homology (53%).
    [Show full text]
  • The Regulatory Region of the Divergent Argecbh Operon in Escherichia Coli K-12
    Volume 10 Number 24 1982 Nucleic Acids Research The regulatory region of the divergent argECBH operon in Escherichia coli K-12 Jacques Piette*, Raymond Cunin*, Anne Boyen*, Daniel Charlier*, Marjolaine Crabeel*, Franpoise Van Vliet*, Nicolas Glansdorff*, Craig Squires+ and Catherine L.Squires + *Nficrobiology, Vrije Universiteit Brussel, and Research Institute of the CERIA, 1, Ave. E. Giyson, B-1070 Brussels, Belgium, and +Department of Biological Sciences, Columbia University, New York, NY 10027, USA Received 24 September 1982; Revised and Accepted 24 November 1982 ABSTRACT The nucleotide sequence of the control region of the divergent argECBH operon has been established in the wild type and in mutants affecting expression of these genes. The argE and argCBH promoters face each other and overlap with an operator region containing two domains which may act as distinct repressor binding sites. A long leader sequence - not involved in attenuation - precedes argCBH. Overlapping of the argCBH promoter and the region involved in ribosome mobilization for argE translation explains the dual effect of some mutations. Mutations causing semi-constitutive expression of argE improve putative promoter sequences within argC. Implications of these results regarding control mechanisms in amino acid biosynthesis and their evolution are discussed. INTRODUCTION Divergently transcribed groups of functionally related genes are not exceptional in Escherichia coli (1-10). In only a few instances, however, are the sites responsible for the expression and the regulation of the flanking genes organized in an inte- grated fashion such that the gene cluster constitutes a bipolar operon, with an internal operator region flanked by promoters facing each other. The argECBH cluster (Fig.1) is one of the earliest reported examples of such a pattern.
    [Show full text]
  • A STAT Protein Domain That Determines DNA Sequence Recognition Suggests a Novel DNA-Binding Domain
    Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press A STAT protein domain that determines DNA sequence recognition suggests a novel DNA-binding domain Curt M. Horvath, Zilong Wen, and James E. Darnell Jr. Laboratory of Molecular Cell Biology, The Rockefeller University, New York, New York 10021 Statl and Stat3 are two members of the ligand-activated transcription factor family that serve the dual functions of signal transducers and activators of transcription. Whereas the two proteins select very similar (not identical) optimum binding sites from random oligonucleotides, differences in their binding affinity were readily apparent with natural STAT-binding sites. To take advantage of these different affinities, chimeric Statl:Stat3 molecules were used to locate the amino acids that could discriminate a general binding site from a specific binding site. The amino acids between residues -400 and -500 of these -750-amino-acid-long proteins determine the DNA-binding site specificity. Mutations within this region result in Stat proteins that are activated normally by tyrosine phosphorylation and that dimerize but have greatly reduced DNA-binding affinities. [Key Words: STAT proteins; DNA binding; site selection] Received January 6, 1995; revised version accepted March 2, 1995. The STAT (signal transducers and activators if transcrip- Whereas oligonucleotides representing these selected se- tion) proteins have the dual purpose of, first, signal trans- quences exhibited slight binding preferences, the con- duction from ligand-activated receptor kinase com- sensus sites overlapped sufficiently to be recognized by plexes, followed by nuclear translocation and DNA bind- both factors. However, by screening different natural ing to activate transcription (Darnell et al.
    [Show full text]
  • On the Prediction of DNA-Binding Preferences of C2H2-ZF Domains Using Structural Models: Application on Human CTCF
    bioRxiv preprint doi: https://doi.org/10.1101/859934; this version posted November 29, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. On the prediction of DNA-binding preferences of C2H2-ZF domains using structural models: application on human CTCF. Alberto Meseguer1,*, Filip Årman1,*, Oriol Fornes2, Ruben Molina1, Jaume Bonet3, Narcis Fernandez-Fuentes4,5, Baldo Oliva1. 1 Structural Bioinformatics Lab (GRIB-IMIM), Department of Experimental and Health Science, University Pompeu Fabra, Barcelona 08005, Catalonia, Spain, 2 Centre for Molecular Medicine and Therapeutics, BC Children's Hospital Research Institute, Department of Medical Genetics, University of British Columbia, Vancouver, BC V5Z 4H4, Canada, 3 Laboratory of Protein Design & Immunoengineering, School of Engineering, Ecole Polytechnique Federale de Lausanne, Lausanne 1015, Vaud, Switzerland, 4 Department of Biosciences, U Science Tech, Universitat de Vic-Universitat Central de Catalunya, Vic 08500, Catalonia, Spain, 5IBERS, Institute of Biological, Environmental and Rural Science, Aberystwyth University U.K *Both authors contributed equally to the paper as first authors. ABSTRACT Cis2-His2 zinc finger (C2H2-ZF) proteins are the largest family of transcription factors in human and higher metazoans. However, the DNA-binding preferences of many members of this family remain unknown. We have developed a computational method to predict these DNA-binding preferences. We combine information from crystal structures composed by C2H2-ZF domains and from bacterial one-hybrid experiments to compute scores for protein-DNA binding based on statistical potentials.
    [Show full text]
  • A History of CRISPR-CAS
    JB Accepted Manuscript Posted Online 22 January 2018 J. Bacteriol. doi:10.1128/JB.00580-17 Copyright © 2018 American Society for Microbiology. All Rights Reserved. 1 2 3 History of CRISPR-Cas from encounter with a mysterious Downloaded from 4 repeated sequence to genome editing technology 5 6 Yoshizumi Ishino,1, 2,* Mart Krupovic,1 Patrick Forterre1, 3 7 http://jb.asm.org/ 8 1Unité de Biologie Moléculaire du Gène chez les Extrêmophiles, Département de 9 Microbiologie, Institut Pasteur, F-75015, Paris, France, 2Department of 10 Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, on March 9, 2018 by UNIV OF COLORADO 11 Fukuoka 812-8581, Japan. 3Institute of Integrative Cellular Biology, Université 12 Paris Sud, 91405 Orsay, Cedex France 13 14 15 Running title: Discovery and development of CRISPR-Cas research 16 17 * Correspondence to 18 Prof. Yoshizumi Ishino 19 Department of Bioscience and Biotechnology, 20 Faculty of Agriculture, Kyushu University, 21 Fukuoka 812-8581, Japan 22 [email protected] 1 23 ABSTRACT 24 CRISPR-Cas systems are well known acquired immunity systems that are 25 widespread in Archaea and Bacteria. The RNA-guided nucleases from Downloaded from 26 CRISPR-Cas systems are currently regarded as the most reliable tools for 27 genome editing and engineering. The first hint of their existence came in 1987, 28 when an unusual repetitive DNA sequence, which subsequently defined as a 29 cluster of regularly interspersed short palindromic repeats (CRISPR), was http://jb.asm.org/ 30 discovered in the Escherichia coli genome during the analysis of genes involved 31 in phosphate metabolism.
    [Show full text]