The structure of the Sleeping Beautytransposase. Sleeping Beautyconsists of paired like DNA binding domain, Nuclear Localization Signal and the C terminal catalytic domain.
A typical SB transposition starts with the binding of the transposase to its IRs followed by the synaptic complex formation in which the two transposon ends are brought proximal together. It is then excised from the donor locus and reintegrates into a new locus which in turn creates a 5 bp foot print mutation in the donor site (Ivics et al., 1997).
Figure 1.5 Schematic representation of the cut-and-paste transposition. The transposase gene (blue box) is flanked by the inverted repeats (IR; grey arrows). The transposase (green circle), the only protein needed for the transposition reaction, binds to the inverted repeats, catalyzes the excision of the transposable element from the donor locus (green lines) and mediates the integration of the element into a new DNA locus (yellow lines). DNA breaks at the integration and donor site are repaired by the host DNA repair machinery.
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The detailed steps are elaborated by a schematic diagram. SB Transposase can recognize IRs either in cis or trans arrangement which makes it possible to physically separate the transposase gene from the IRs. The trans arrangement makes it easy to clone any gene of interest between the IRs (Figure ) with the transposase driven by a strong promoter will thus serve as a valuable for genome engineering. But the efficiency of transposition reaction as a two component system can be limited by a phenomenon termed overproduction inhibition which is wide spread in Tc1/mariner family & the cargo capacity of cloned DNA insert between the IRs (Grabundzija et al., 2010).
Figure 1.6 The Sleeping Beautytransposon system. (A) Natural arrangement of the Sleeping Beautytransposon. The transposase gene (blue box) is flanked by the inverted repeats (IR; grey arrows) that contain the transposase binding sites (DR; white arrows). (B) Laboratory arrangement of the Sleeping Beautygene transfer vector system. The transposase coding region is replaced by a gene of interest (green box). The transposase is provided on a separate plasmid vector expressed from a suitable promoter (blue arrow).
Systematic study for the most efficient transposon vector system among Tc1/mariner family like Tc1, Tc3, Himar1 and Mos1 with invitro mammalian cell culture assay determined that SB is the most efficient system(Fischer et al., 2001). SB has a target site preference for palindromic AT-repeat, ATATATAT, in which the central TA is the canonical target site & upon transposition undergoes TA dinucleotide duplication in the target site repaired by host repair machinery. It is the most active transposon system of Tc1/Mariner family used in genome manipulation and various gene therapy applications in vertebrates (reviewed in Mátés et al., 2007). Molecular reconstruction by in vitro evolution lead to the generation of novel hyperactive forms of SB in which, SB100x is the most active and supported 35-50% stable gene transfer in human primary cells & 45% stable transgenesis in mouse zygotes (Mátés et al., 2009)
1.1 Host Factors & Sleeping Beautyregulation
SB transposon has wide range of activity in vertebrates with different efficiency & among cells of different tissues of the same species. Possible explanation for such difference in efficiency can be attributed to the interaction of the transposition machinery with host factors. Nevertheless, if host proteins do indeed participate in the transposition reaction, they must be conserved in vertebrates. A highly conserved DNA-bending protein belonging to the high-mobility group of proteins, HMGB1, was first identified as a cofactor of SB transposition necessary for transposes-transposon complexes at the internal DRs (Zayed, 2003). Transposition of SB leads to DNA double strand breaks, which are shown to be repaired by host repair machinery.Ku70, an important protein involved in non-homologous end joining repair pathway interacts physically with SB transposase, establishing a functional link between the host DNA repair machinery and transposase.(Izsvák et al., 2004).
The epigenetic modification of SB transposable element by CpG methylation within the transposon sequence enhances the transposition frequency of the SB transposon (Yusa et al., 2004). SB, by its interaction with another host encoded protein Miz-1 a transcription factor down-regulates cyclin D1 expression in human cells and induces G1 slowdown, which can be seen as selfish act for maximal transpositional event (Walisko et al., 2006).
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1.2 HMG2L1 induces transcription of the transposon 5â€²-UTR
The high-mobility-group-box (HMGB) proteins are one of the three HMG chromosomal protein super families which can be further classified into two major subgroups: Group 1 proteins have more than one HMGB domain with a long acidic C-terminal tail without particular sequence specificity. Group 2 proteins contain only a single HMGB domain with some degree of sequence specificity and can act as a transcriptional factor (Bustin, 1999). It is important to note that HMG2L1 belongs to the second subgroup of HMGB proteins. It has been shown that HMG2L1 negatively regulates Wnt signaling by interacting with a novel NLK-binding protein (Yamada et al., 2003). Further studies has shown its role in attenuating smooth muscle differentiation (Zhou et al., 2010). In search of other host proteins by yeast two hybrid screen that can possibly interact with SB transposase, resulted in another high mobility protein HMG2L1 (high-mobility group protein 2-like 1Â¬Â¬).This interacts with SB transposase and then binds on it's 5' untranslated region thereby, driving its expression although, in the presence of SB transposase, the HMG2L1 is negatively regulated by feedback inhibition (Walisko et al., 2008).
1.3 Regulation of transcription factor activity by SUMO modification
SUMO proteins belong to a family of small proteins which are structurally related to ubiquitin and can be covalently attached to lysine residues within its target proteins (Gill, 2004). SUMOylation is a post-translational modification involved in various cellular processes such as nuclear cytosolic transport, transcriptional regulation, protein stability and progression through the cell cycle (Johnson, 2004). In SUMOylation, the target lysine generally falls within a recognizable consensus, namely Ïˆ-Lys-X-Glu (where Ïˆ is a large hydrophobic amino acid, most commonly isoleucine or valine, and X is any residue) (Mahajan et al., 1997; Melchior, 2000). In addition to the covalent attachment of SUMO to lysine residues in target proteins, a recent work has shown a class of SUMO-interacting motifs (SIMs) that mediate non-covalent interactions with SUMO (Minty et al., 2000; Song et al., 2004). An SXS triplet motif in the protein might be crucial for SUMO interaction (Minty et al., 2000) but this motif was proved not to be correct and the best characterized SUMO interaction motifs (SIMs) normally have the consensus sequence, V/I-x-V/I-V/I or V/I-V/I-x-V/I/L, where position two or three can be any amino acid (Song et al., 2004). Three SUMO family members SUMO1/Smt3C, SUMO2/Smt3A and SUMO3/Smt3B, are known to exist in mammals (Hay, 2005). Like ubiquitin, SUMO2 and SUMO3 have been shown to have the ability to form polymeric chains, suggesting that modification by SUMO1, SUMO2 or SUMO3 might have distinct functional consequences (Saitoh and Hinchey, 2000; Tatham et al., 2001). PIAS proteins were first shown as protein inhibitors of activated STAT (signal transducer and activator of transcription) (Liu et al., 1998) but further studies on its RING finger motif revealed similar kind of function like RING-type ubiquitin E3 ligases and confirmed its function as SUMO E3 ligase for SUMO modification (Sachdev et al., 2002). But PIAS proteins bearing mutations in the RING domain suggest that the PIAS proteins have both E3 SUMO ligase-dependent and -independent activities (Gross et al., 2004). SUMOylation is a highly dynamic process enabling transient responses to be elicited which is controlled by a group of two competing conjugating and de-conjugating enzymes, the latter by SENP [SUMO1/sentrin/SMT3 (suppressor of mif two 3 homologue 1)-specific peptidase] family of SUMO-specific proteases (Witty et al., 2010).
Figure 1.7 The regulation of sumoylation pathway - SUMO is synthesized as a precursor and processed by hydrolase to expose the carboxy-terminal double-glycine motif available for conjugation. (A) SUMO conjugation to target substrates requires an enzymatic cascade that involves three classes of enzymes (E1 â†’ E2 â†’ E3). The sentrin/SUMOspecific proteases (SENPs) are responsible for the deconjugation pathway as well as the maturation process of newly synthesized SUMO proteins. Figure redrawn from (Woo & Abe, 2010). (B) Primary domain structure of PIAS1- PIAS family of proteins contains a putative zinc-binding motif and a highly acidic region but the presence of unique SP-RING domain that execute sumoylation by enzymatic process. Figure redrawn from (Woo & Abe, 2010)
SUMO modification on a protein can influence the molecular properties of a protein in different ways. Indeed, studies have shown that SUMO can affect protein-protein interactions, protein-DNA interactions, subcellular nuclear localization and can act as an antagonist of ubiquitin (Verger et al., 2003). The SUMOylation of transcription factors have been shown to have various effects on their activity. For example, SUMOylation of the heat shock factors HSF1 and HSF2 can upregulate their DNA binding activity (Goodson et al., 2001; Hong et al., 2001). In contrast, the SUMOylation of many diverse transcription factors, such as Sp3, c-Jun, c-Myb, AP2 (activating enhancer-binding protein-2) and nuclear receptors, has been shown to correlate with down-regulation of their transcriptional activation potency (Bies et al., 2002; Eloranta and Hurst, 2002; Muller et al., 2000; Ross et al., 2002).
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Figure 1.8 Effect of SUMO modification on the protein. Some of the known functions of SUMOylation are indicated with respect to transcriptional regulation.
1.4 SB based genome manipulations
The inherent capacity of transposons to hop within the genome makes it an excellent gene vector for genome manipulations. SB has been exclusively used for transposon based genetic strategies in vertebrate genomes for somatic & germ line transgenesis and as insertional mutagenesis for both forward and reverse genetic screens (Ivics et al., 2009). This study aimed to highlight the application of SB 100x in mouse and rodent transgenesis & targeted transgene combined with recombination mediated cassette exchange (RMCE). For detailed information on transposon based genome manipulation, one can refer the review of (Mátés et al., 2007). Targeting transgenes by transposon has a number of advantages compared to conventional methods.
A single copy of DNA construct can be inserted into a genome location that allows the desired developmental and tissue specific expression of a transgene whereas, traditional pronuclear injection with the linearized DNA leads to concatemer formation of transgene resulting in silencing (Mátés, 2011).
Transposons do not have any target preference in the genome and this gives the possibility to scan the genome and fish out safe locus for transgene expression. It can be combined with the concept of recombinase-mediated cassette exchange to perform site-specific chromosomal integration followed by exchange with the desired transgene (Osborne et al., 1995). .
The concept is schematically explained below & efforts are undertaken to create a model of mouse or rat combining the concept transposon based RMCE.
Figure 1.9 Schematic depiction of transposon based recombination mediated cassette exchange (RMCE) to create transposons-tagged genomic loci, wherein a transgene of interest can be exchanged.
Transposon based genetic strategies were recently established in vertebrates and a fast and cutting edge progress in this field indicates that transposable elements will indeed serve as indispensable tools in the genetic toolkit for vertebrate models.