Mechanism and Regulation of P Element Transposition

P elements were first discovered in the fruit fly Drosophila melanogaster as the causative agents of a syndrome of aberrant genetic traits called hybrid dysgenesis. This occurs when P element-carrying -males mate with females that lack P elements and results in progeny displaying sterility, mutations and chromosomal rearrangements. Since then numerous genetic, developmental, biochemical and structural studies have culminated in a deep understanding of P element transposition: from the cellular regulation and repression of transposition to the mechanistic details of the transposase nucleoprotein complex. Recent studies have revealed how piwi-interacting small RNA pathways can act to control splicing of the P element pre-mRNA to modulate transposase production in the germline. A recent cryo-electron microscopy structure of the P element transpososome reveals an unusual DNA architecture at the transposon termini and shows that the bound GTP cofactor functions to position the transposon ends within the transposase active site. Genome sequencing efforts have shown that there are P element transposase-homologous genes (called THAP9) in other animal genomes, including humans). This review highlights recent and previous studies, which together have led to new insights, and surveys our current understanding of the biology, biochemistry, mechanism and regulation of P element transposition.Human Frontier Science Program (CDA-00032/2018

P element transposases in Drosophila melanogaster and other Eukaryotes

Transposable elements are mobile genetic sequences that are found in the genomes of nearly all organisms. DNA transposons constitute a major class of transposable elements and can move throughout the host genome by a cut-and-paste mechanism catalyzed by an encoded transposase protein. Although transposase activity can be detrimental to the host, numerous examples of host benefit have been documented. Over evolutionary time transposon-related sequences and proteins have been adapted to serve a wide range of cellular functions, a process termed transposon domestication.The Drosophila P element is one well-studied example of a eukaryotic DNA transposable element. Although the encoded P element transposase protein has been biochemically characterized, it exhibits several features that distinguish it from the other characterized DNA transposases. Namely, P element transposase requires a guanosine triphosphate (GTP) cofactor and generates unusually long 17 nucleotide staggered DNA breaks at the transposon ends during transposition. To gain insight into the molecular basis of these distinguishing features we determined the cryo-EM structure of the Drosophila P element transposase strand transfer complex (STC) to 3.6 Å - a nucleoprotein complex in which the transposase protein is bound to P element donor DNAs covalently joined to a target DNA. Our structure reveals that the STC is dimeric, the P element donor DNAs adopt a highly unusual DNA geometry and further reveals a function for GTP in positioning the P element ends into the transposase active site for catalysis. This structure provides the first view of the P element superfamily of eukaryotic DNA transposases, offers new insights in P element transposition and implies a transposition pathway that is mechanistically distinct from other cut-and-paste DNA transposases.Furthermore, bioinformatic and biochemical analysis have identified C2CH DNA binding domain termed the THAP domain. This novel and evolutionarily conserved domain is found across a wide range of animal genomes, including vertebrates, invertebrates, Drosophila P element transposase, in primates and in 12 human genes. Of the 12 THAP domain containing genes in humans, THAP9 is homologous to the entirety of Drosophila P element transposase, still has DNA transposase activity, but lacks the hallmarks of an active DNA transposable element. maintained. The evidence implies that THAP9 has likely been domesticated/adapted by the cell in early chordates from an ancient THAP9-like P element transposon, such as those found in Ciona. However, a cellular function for THAP9 has not been identified. In an attempt to elucidate a cellular function for THAP9, we carried out genome-editing in human embryonic stem cells (hESCs) to either knockout or epitope tag the endogenous THAP9 gene. Disruption of THAP9 did not produce overt phenotypic changes in hESCs and did not affect differentiation into fibroblast-like cells, indicating that THAP9 is likely not required for the hESC maintenance. However, endogenously epitope tagged THAP9 is translated, can be immunoprecipitated and localizes to the nucleus in hESCs. To determine potential THAP9 human genome cleavage and binding sites, we raised an antibody to purified, recombinant human THAP9 protein, performed direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing, or BLESS, to detect potential DNA cleavage site, a method used successfully to find Cas9 off-target genomic cleavage sites and ChIP-Nexus experiment, a chromatin immunoprecipitation method similar to ChIP-Exo. The ongoing analysis and comparison of both the BLESS and ChIP-Nexus sequencing data should identify genomic binding sites, potential genomic DNA cleavage sites, motifs associated with human THAP9 DNA binding and cleavage and should uncover a cellular function for the human THAP9 gene.While these projects are essentially independent of one another, they all relate to P element DNA transposases. Together, they hopefully contribute to a deeper understanding of the mechanisms of P element transposition and the expanding roles that transposase-related proteins play in the context of cellular function in human cells

P element transposases in Drosophila melanogaster and other Eukaryotes

Transposable elements are mobile genetic sequences that are found in the genomes of nearly all organisms. DNA transposons constitute a major class of transposable elements and can move throughout the host genome by a cut-and-paste mechanism catalyzed by an encoded transposase protein. Although transposase activity can be detrimental to the host, numerous examples of host benefit have been documented. Over evolutionary time transposon-related sequences and proteins have been adapted to serve a wide range of cellular functions, a process termed transposon domestication.The Drosophila P element is one well-studied example of a eukaryotic DNA transposable element. Although the encoded P element transposase protein has been biochemically characterized, it exhibits several features that distinguish it from the other characterized DNA transposases. Namely, P element transposase requires a guanosine triphosphate (GTP) cofactor and generates unusually long 17 nucleotide staggered DNA breaks at the transposon ends during transposition. To gain insight into the molecular basis of these distinguishing features we determined the cryo-EM structure of the Drosophila P element transposase strand transfer complex (STC) to 3.6 Å - a nucleoprotein complex in which the transposase protein is bound to P element donor DNAs covalently joined to a target DNA. Our structure reveals that the STC is dimeric, the P element donor DNAs adopt a highly unusual DNA geometry and further reveals a function for GTP in positioning the P element ends into the transposase active site for catalysis. This structure provides the first view of the P element superfamily of eukaryotic DNA transposases, offers new insights in P element transposition and implies a transposition pathway that is mechanistically distinct from other cut-and-paste DNA transposases.Furthermore, bioinformatic and biochemical analysis have identified C2CH DNA binding domain termed the THAP domain. This novel and evolutionarily conserved domain is found across a wide range of animal genomes, including vertebrates, invertebrates, Drosophila P element transposase, in primates and in 12 human genes. Of the 12 THAP domain containing genes in humans, THAP9 is homologous to the entirety of Drosophila P element transposase, still has DNA transposase activity, but lacks the hallmarks of an active DNA transposable element. maintained. The evidence implies that THAP9 has likely been domesticated/adapted by the cell in early chordates from an ancient THAP9-like P element transposon, such as those found in Ciona. However, a cellular function for THAP9 has not been identified. In an attempt to elucidate a cellular function for THAP9, we carried out genome-editing in human embryonic stem cells (hESCs) to either knockout or epitope tag the endogenous THAP9 gene. Disruption of THAP9 did not produce overt phenotypic changes in hESCs and did not affect differentiation into fibroblast-like cells, indicating that THAP9 is likely not required for the hESC maintenance. However, endogenously epitope tagged THAP9 is translated, can be immunoprecipitated and localizes to the nucleus in hESCs. To determine potential THAP9 human genome cleavage and binding sites, we raised an antibody to purified, recombinant human THAP9 protein, performed direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing, or BLESS, to detect potential DNA cleavage site, a method used successfully to find Cas9 off-target genomic cleavage sites and ChIP-Nexus experiment, a chromatin immunoprecipitation method similar to ChIP-Exo. The ongoing analysis and comparison of both the BLESS and ChIP-Nexus sequencing data should identify genomic binding sites, potential genomic DNA cleavage sites, motifs associated with human THAP9 DNA binding and cleavage and should uncover a cellular function for the human THAP9 gene.While these projects are essentially independent of one another, they all relate to P element DNA transposases. Together, they hopefully contribute to a deeper understanding of the mechanisms of P element transposition and the expanding roles that transposase-related proteins play in the context of cellular function in human cells

Mechanism and Regulation of P Element Transposition

Peer reviewed: TrueP elements were first discovered in the fruit fly Drosophila melanogaster as the causative agents of a syndrome of aberrant genetic traits called hybrid dysgenesis. This occurs when P element-carrying -males mate with females that lack P elements and results in progeny displaying sterility, mutations and chromosomal rearrangements. Since then numerous genetic, developmental, biochemical and structural studies have culminated in a deep understanding of P element transposition: from the cellular regulation and repression of transposition to the mechanistic details of the transposase nucleoprotein complex. Recent studies have revealed how piwi-interacting small RNA pathways can act to control splicing of the P element pre-mRNA to modulate transposase production in the germline. A recent cryo-electron microscopy structure of the P element transpososome reveals an unusual DNA architecture at the transposon termini and shows that the bound GTP cofactor functions to position the transposon ends within the transposase active site. Genome sequencing efforts have shown that there are P element transposase-homologous genes (called THAP9) in other animal genomes, including humans). This review highlights recent and previous studies, which together have led to new insights, and surveys our current understanding of the biology, biochemistry, mechanism and regulation of P element transposition.Human Frontier Science Program (CDA-00032/2018

Mechanism and regulation of P element transposition.

P elements were first discovered in the fruit fly Drosophila melanogaster as the causative agents of a syndrome of aberrant genetic traits called hybrid dysgenesis. This occurs when P element-carrying males mate with females that lack P elements and results in progeny displaying sterility, mutations and chromosomal rearrangements. Since then numerous genetic, developmental, biochemical and structural studies have culminated in a deep understanding of P element transposition: from the cellular regulation and repression of transposition to the mechanistic details of the transposase nucleoprotein complex. Recent studies have revealed how piwi-interacting small RNA pathways can act to control splicing of the P element pre-mRNA to modulate transposase production in the germline. A recent cryo-electron microscopy structure of the P element transpososome reveals an unusual DNA architecture at the transposon termini and shows that the bound GTP cofactor functions to position the transposon ends within the transposase active site. Genome sequencing efforts have shown that there are P element transposase-homologous genes (called THAP9) in other animal genomes, including humans. This review highlights recent and previous studies, which together have led to new insights, and surveys our current understanding of the biology, biochemistry, mechanism and regulation of P element transposition

Structure of a P element transposase–DNA complex reveals unusual DNA structures and GTP-DNA contacts

P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the Drosophila P element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprotein complex reveals that the terminal single-stranded transposon DNA adopts unusual A-form and distorted B-form helical geometries that are stabilized by extensive protein-DNA interactions. Additionally, we infer that the bound guanosine triphosphate cofactor interacts with the terminal base of the transposon DNA, apparently to position the P element DNA for catalysis. Our structure provides the first view of the P element transposase superfamily, offers new insights into P element transposition and implies a transposition pathway fundamentally distinct from other cut-and-paste DNA transposases