Chapter 38 : Sleeping Beauty Transposition

The SB transposon system. (A) Structure of the SB 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 SB. 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 Current Gene Therapy ( 161 ) with permission from the publisher.

Structural and functional components of SB. 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 Molecular Therapy ( 164 ) with permission from the publisher.

Structures of the PAI subdomain of the SB transposase and the DNA-bound N-terminal DNA-binding subdomains of the Tc3 and Mos1 mariner transposases and the Pax5 transcription factor. Residues on the second and third alpha-helices of the SB PAI subdomain are directly involved in DNA-binding. Reprinted from Protein Science ( 13 ) with permission from the publisher.

Comparison of different hyperactive versions of the SB 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 SB transposase and with the SB100X hyperactive variant.

Mechanism and regulation of SB 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.

The UTRs of the SB transposon exhibit moderate, directional promoter activities. (A) Transcriptional activities residing within the SB 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 SB; green box: right IR of SB; beige box: left IR of Frog Prince; black lines connecting the IRs and the luciferase gene represent transposon sequences directly upstream of the transposase coding regions. The 5′-UTR of SB can drive transposase expression at a level sufficient for the detection of chromosomal transposition events in cultured cells. A neo-tagged SB transposon plasmid was cotransfected together with an SB 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 SB 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 SB transposase downregulates transcription from the 5′-UTR. Reprinted from Molecular Therapy ( 54 ) with permission from the publisher.

A model for the role of HMGB1 in SB synaptic complex formation. SB 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 Nucleic Acids Res ( 53 ) with permission from the publisher.

A model for the enhancing effect of a compact chromatin structure on SB 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 Mobile DNA ( 73 ) with permission from the publisher.

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 CMLS ( 165 ) with permission from the publisher.

The SB transposase modulates cell-cycle progression through interaction with Miz-1. The SB 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 PNAS ( 103 ) with permission from the publisher.

Genomic insertion preferences of SB. (A) Consensus sequence of SB insertion sites. Seqlogo analysis and nucleotide probability plot of SB 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 Molecular Therapy ( 126 ) with permission from the publisher. Part (B) reprinted from BioEssays ( 170 ) with permission from the publisher.

Broad applicability of SB transposon-based gene vectors in vertebrate genetics. Reprinted from Mobile DNA ( 168 ) with permission from the publisher.