Unraveling the Transposition Mechanism of PiggyBac Transposon and Its Potential Application on Site-Specific Genome Targeting

Transposons are mobile DNA elements that move within its host genome and sometimes between genomes through a horizontal transfer. Forty-four percent of the human genome consists of the transposon-like repetitive element, indicating that the possible interplay between transposon and host genome may provide the driven force for genome evolution. The intimate interaction between a transposon and its host requires mechanisms that constrain the potential lesion imposing upon the host genome during transposition while still allow the minimal occurrence of transposition to generate a “competent” genome in a versatile environment. It is conceivable that the host factors and transposon itself play a major role in these regulatory mechanisms to reach the goal of mutually benefiting the fitness for both sides. Therefore, uncovering the host factors interacting with transposons shall shed light on the mechanisms for regulating the transposition of transposons as well as shaping the genome during the evolution course. DNA transposons, constituting 3% of the human genome, jump in and out of the host genome in a cut-and-paste fashion. Among various DNA transposons, piggyBac transposon, isolated from cabbage looper moth, displays highly efficient transposition activity across different phyla, including human and mice. Although the human genome exists approximate 2,000 recognizable endogenous piggyBac-like elements, namely MER85 and MER75, these elements may be relics of human genome evolution since no mobilization of these elements are detected. Nevertheless, the significant difference in transposition activities of piggyBac observed in various host genomes indicated the presence of host regulatory mechanisms governing its transposition activity. And these observations make piggyBac transposon an ideal model transposon system to unravel the basic molecular mechanisms of DNA transposition as well as the regulatory mechanism co-evolved with its host for genome evolution. Recently, piggyBac transposon has leaped as one of the most attractive nonviral-based genetic tools for mammalian gene transfer. However, advancing piggyBac to clinical applications remains distant, largely due to the lack of knowledge on how its transposition is regulated. Thereby, in this research proposal, I will take an initiative to address this central issue by identifying host factors interacting with piggyBac transposase and its terminal inverted repeats (TIRs) and performing function assays to reveal the biological significance of the interaction. Findings derived from this proposed study will provide invaluable bases for developing the piggyBac-based platform capable of performing site-specific transgene targeting effectively, a system receiving enormous attention from the field of mammalian transgenesis and gene therapy. To achieve this ultimate goal, here we would also take the initiative by adopting the TALE technology to generate TALE- piggyBac transposases recognizing a predefined site on the endogenous HPRT locus, and then testing its ability in site-specific targeting in the presence or absence of those host factors. In sum, potential findings from this research proposal not only will shed lights on revealing the molecular mechanism regarding how the genome stability for homeostasis compromises with the genome modification for adapting the versatile environments to shape the contemporary mammalian genome, but also will facilitate the advancement of piggyBac transposon as a safe, highly effective, and off-the-shelf genetic tools for mammalian transgenesis and clinical applications, such as gene therapy.

Project ID:PC10207-0344
External Project ID:NSC102-2320-B182-034