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Transposition

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  • Authors: Zoltán Ivics1, Zsuzsanna Izsvák2
  • Editors: Mick Chandler3, Nancy Craig4
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany; 2: Max Delbrück Center for Molecular Medicine, Berlin, Germany; 3: Université Paul Sabatier, Toulouse, France; 4: Johns Hopkins University, Baltimore, MD
  • Source: microbiolspec March 2015 vol. 3 no. 2 doi:10.1128/microbiolspec.MDNA3-0042-2014
  • Received 10 July 2014 Accepted 14 August 2014 Published 05 March 2015
  • Zoltán Ivics, zoltan.ivics@pei.de and Zsuzsanna Izsvák, zizsvak@mdc-berlin.de
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  • Abstract:

    () is a synthetic transposon that was constructed based on sequences of transpositionally inactive elements isolated from fish genomes. is a Tc1/ superfamily transposon following a cut-and-paste transpositional reaction, during which the element-encoded transposase interacts with its binding sites in the terminal inverted repeats of the transposon, promotes the assembly of a synaptic complex, catalyzes excision of the element out of its donor site, and integrates the excised transposon into a new location in target DNA. transposition is dependent on cellular host factors. Transcriptional control of transposase expression is regulated by the HMG2L1 transcription factor. Synaptic complex assembly is promoted by the HMGB1 protein and regulated by chromatin structure. transposition is highly dependent on the nonhomologous end joining (NHEJ) pathway of double-strand DNA break repair that generates a transposon footprint at the excision site. Through its association with the Miz-1 transcription factor, the transposase downregulates cyclin D1 expression that results in a slowdown of the cell-cycle in the G1 phase, where NHEJ is preferentially active. Transposon integration occurs at TA dinucleotides in the target DNA, which are duplicated at the flanks of the integrated transposon. shows a random genome-wide insertion profile in mammalian cells when launched from episomal vectors and “local hopping” when launched from chromosomal donor sites. Some of the excised transposons undergo a self-destructive autointegration reaction, which can partially explain why longer elements transpose less efficiently. became an important molecular tool for transgenesis, insertional mutagenesis, and gene therapy.

  • Citation: Ivics Z, Izsvák Z. 2015. Transposition. Microbiol Spectrum 3(2):MDNA3-0042-2014. doi:10.1128/microbiolspec.MDNA3-0042-2014.

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/content/journal/microbiolspec/10.1128/microbiolspec.MDNA3-0042-2014
2015-03-05
2017-02-25

Abstract:

() is a synthetic transposon that was constructed based on sequences of transpositionally inactive elements isolated from fish genomes. is a Tc1/ superfamily transposon following a cut-and-paste transpositional reaction, during which the element-encoded transposase interacts with its binding sites in the terminal inverted repeats of the transposon, promotes the assembly of a synaptic complex, catalyzes excision of the element out of its donor site, and integrates the excised transposon into a new location in target DNA. transposition is dependent on cellular host factors. Transcriptional control of transposase expression is regulated by the HMG2L1 transcription factor. Synaptic complex assembly is promoted by the HMGB1 protein and regulated by chromatin structure. transposition is highly dependent on the nonhomologous end joining (NHEJ) pathway of double-strand DNA break repair that generates a transposon footprint at the excision site. Through its association with the Miz-1 transcription factor, the transposase downregulates cyclin D1 expression that results in a slowdown of the cell-cycle in the G1 phase, where NHEJ is preferentially active. Transposon integration occurs at TA dinucleotides in the target DNA, which are duplicated at the flanks of the integrated transposon. shows a random genome-wide insertion profile in mammalian cells when launched from episomal vectors and “local hopping” when launched from chromosomal donor sites. Some of the excised transposons undergo a self-destructive autointegration reaction, which can partially explain why longer elements transpose less efficiently. became an important molecular tool for transgenesis, insertional mutagenesis, and gene therapy.

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FIGURE 1

The transposon system. (A) Structure of the transposon. The central transposase gene (purple box) is flanked by terminal IRs (black arrows) that contain binding sites for the transposase (white arrows). (B) Gene transfer vector system based on . The transposase coding region can be replaced by a gene of interest (yellow box) within the transposable element. This transposon can be mobilized if a transposase source is provided in cells; for example, the transposase can be expressed from a separate plasmid vector containing a suitable promoter (black arrow). Reprinted from ( 161 ) with permission from the publisher. doi:10.1128/microbiolspec.MDNA3-0042-2014.f1

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FIGURE 2

Structural and functional components of . On top, a schematic drawing of the transposon is shown. The terminal IR/DR (black arrows) contain two binding sites for the transposase (white arrows). The element contains a single gene encoding the transposase (purple box). The transposase has an N-terminal, bipartite, paired-like DNA-binding domain containing a GRRR AT-hook motif, an NLS, and a C-terminal catalytic domain. The DNA binding domain consists of a PAI and a RED subdomain containing helix-turn-helix DNA-binding motifs. The DDE amino acid triad is a characteristic signature of the catalytic domain that catalyzes the DNA cleavage and joining reactions. Reprinted from ( 164 ) with permission from the publisher. doi:10.1128/microbiolspec.MDNA3-0042-2014.f2

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FIGURE 3

Structures of the PAI subdomain of the transposase and the DNA-bound N-terminal DNA-binding subdomains of the Tc3 and Mos1 transposases and the Pax5 transcription factor. Residues on the second and third alpha-helices of the PAI subdomain are directly involved in DNA-binding. Reprinted from ( 13 ) with permission from the publisher. doi:10.1128/microbiolspec.MDNA3-0042-2014.f3

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FIGURE 4

Comparison of different hyperactive versions of the transposase in transfected human HeLa cells. The chart shows the respective potential of transposase mutants to generate antibiotic-resistant cell colonies in human cell culture. The Petri dishes on the right show stained, antibiotic-resistant cell colonies obtained with the original transposase and with the 100X hyperactive variant. doi:10.1128/microbiolspec.MDNA3-0042-2014.f4

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FIGURE 5

Mechanism and regulation of transposition. The transposable element consists of a gene encoding a transposase (orange box) bracketed by terminal IRs (solid black arrows) that contain binding sites of the transposase (white arrows) and flanking donor DNA (blue boxes). Transcriptional control elements in the 5′-UTR of the transposon drive transcription (arrow) of the transposase gene. The transposase (purple spheres) binds to its sites within the transposon IRs. Excision takes place in a synaptic complex, and separates the transposon from the donor DNA. The excised element integrates into a TA site in the target DNA (green box) that is duplicated and flanks the newly integrated transposon. On the right, the various steps of transposition are shown. On the left, mechanisms and host factors regulating each step of the transposition reaction are indicated. Reprinted from ( 57 ) with permission from the publisher. doi:10.1128/microbiolspec.MDNA3-0042-2014.f5

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FIGURE 6

The UTRs of the transposon exhibit moderate, directional promoter activities. (A) Transcriptional activities residing within the transposon. On top, a schematic drawing of the transposon is shown. The terminal IRs contain two binding sites for the transposase (white arrows). The element contains a single gene encoding the transposase (purple box). Relative promoter activities as determined by transient luciferase assays in HeLa cells. Activity of a minimal promoter (TATA-box) control was arbitrarily set to value 1. Transposon sequences flanking the transposase gene were placed in front of a luciferase reporter gene in two possible orientations (in the case of the 5′-UTR, the luciferase gene precisely replaces the transposase coding region). Blue box: left IR of ; green box: right IR of ; beige box: left IR of ; black lines connecting the IRs and the luciferase gene represent transposon sequences directly upstream of the transposase coding regions. The 5′-UTR of can drive transposase expression at a level sufficient for the detection of chromosomal transposition events in cultured cells. A neo-tagged transposon plasmid was cotransfected together with an expression construct, in which the transposase is expressed from the 5′-UTR of the transposon or with an empty cloning vector. The difference in numbers of G418-resistant cell colonies is evidence for transposition. (B) A model for transcriptional regulation of the transposase gene. In the wild-type, natural transposon, the central transposase gene (purple box) is flanked by UTRs that include the left and right inverted repeats (IRs, blue and green boxes, respectively) that contain binding sites for the transposase (white arrows). Arrows indicate the direction of transcription that is initiated within the UTRs. HMG2L1 upregulates, whereas transposase downregulates transcription from the 5′-UTR. Reprinted from ( 54 ) with permission from the publisher. doi:10.1128/microbiolspec.MDNA3-0042-2014.f6

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FIGURE 7

A model for the role of HMGB1 in synaptic complex formation. transposase (pink spheres) recruits HMGB1 (dotted hexagons) to the transposon IRs. First, HMGB1 stimulates specific binding of the transposase to the inner binding sites (IDRs). Once in contact with DNA, HMGB1 bends the spacer regions between the DRs, thereby assuring correct positioning of the outer sites (ODRs) for binding by the transposase. Cleavage (scissors) proceeds only if complex formation is complete. The complex includes the four binding sites (black boxes) and a tetramer of the transposase. Reprinted from ( 53 ) with permission from the publisher. doi:10.1128/microbiolspec.MDNA3-0042-2014.f7

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FIGURE 8

A model for the enhancing effect of a compact chromatin structure on transposition. Euchromatin contains DNA wrapped around nucleosomes in a “beads-along-a-string”-like conformation (upper panel). Transposase subunits bound within the transposon IRs are separated by 166 bp DNA. Heterochromatin (lower panel), characterized by DNA CpG methylation and specific histone tail modifications, e.g., trimethylated lysine 9 of histone H3, features a higher histone : DNA ratio. Positioning of a nucleosome between the transposase binding sites (small orange arrows) shortens the distance between these sites, thereby facilitating the formation of a transposase dimer per IR and subsequent assembly of the synaptic complex. Reprinted from ( 73 ) with permission from the publisher. doi:10.1128/microbiolspec.MDNA3-0042-2014.f8

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FIGURE 9

Molecular events during cut-and-paste transposition. The transposase initiates the excision of the transposon with staggered cuts and reintegrates it at a TA target dinucleotide. The single-stranded gaps at the integration site as well as the double-strand DNA breaks in the donor DNA are repaired by the host DNA repair machinery. After repair, the target TA is duplicated at the integration site, and a small footprint is left behind at the site of excision. The footprint is generated by the NHEJ pathway of DSB repair, and the central A:A mismatch is likely repaired by the mismatch repair system of the cell. Reprinted from ( 165 ) with permission from the publisher. doi:10.1128/microbiolspec.MDNA3-0042-2014.f9

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FIGURE 10

The transposase modulates cell-cycle progression through interaction with Miz-1. The transposase, through its interaction with Miz-1, downregulates cyclin D1 expression, which results in an inhibition of the G1/S transition of the cell-cycle. Reprinted from ( 103 ) with permission from the publisher. doi:10.1128/microbiolspec.MDNA3-0042-2014.f10

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FIGURE 11

Genomic insertion preferences of . (A) Consensus sequence of insertion sites. Seqlogo analysis and nucleotide probability plot of insertion sites in HeLa cells. Twenty base pairs upstream and downstream of the TA target sites were analyzed. The y-axis represents the strength of the information, with 2 bits being the maximum for a DNA sequence. (B) Relative frequencies of insertions into genes by retroviruses and transposons. The top portions of the graphs indicate an over-representation of genic insertions as compared to random. Part (A) reprinted from ( 126 ) with permission from the publisher. Part (B) reprinted from ( 170 ) with permission from the publisher. doi:10.1128/microbiolspec.MDNA3-0042-2014.f11

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FIGURE 12

Broad applicability of transposon-based gene vectors in vertebrate genetics. Reprinted from ( 168 ) with permission from the publisher. doi:10.1128/microbiolspec.MDNA3-0042-2014.f12

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