Functional indications for transposase domestications – Characterization of the human piggyBac transposase derived (PGBD) activities

Kolacsek, Orsolya; Wachtl, Gerda; Fóthi, Ábel; Schamberger, Anita; Sándor, Sára; Pergel, Enikő; Varga, Nóra; Raskó, Tamás; Izsvák, Zsuzsanna; Apáti, Ágota; Orbán, Tamás I.

doi:10.1016/j.gene.2022.146609

Cited by 5 publications

(13 citation statements)

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“…Seeing that the NTDR is necessary for the proper function of mPB, we asked whether the presence of such a longer disordered region at the N-terminus is a characteristic feature of other piggyBac -related proteins. Using the IUPred2 program [ 45 , 46 ] to predict the disorder tendencies of the five domesticated human piggyBac -derived sequences (PGBD1-5) [ 22 , 31 ], we found that all of these proteins contain a long stretch of structurally disordered regions upstream of the transposase domains, even PGBD1, which has gained additional N-terminal protein domains during evolution ( Figure 5 A). Inspired by this, we conducted a further systematic analysis of all the currently available sequences of this superfamily of DNA transposons.…”

Section: Resultsmentioning

confidence: 99%

“…Our bioinformatic analyses provided evidence that such a disordered protein region is present at the N-terminal part of the transposase domain in other piggyBac -related proteins. It was revealed that the position of this region is well-conserved among the members of the piggyBac superfamily, even among the domesticated proteins that were shown to have lost their ability for transposition, including the five domesticated human PGBD proteins [ 22 ]. It is intriguing that regardless of the acquisition of other non-transposase domains during evolution (as in the case of PGBD1 or PGBD4), the position of the disordered region is strictly kept upstream of the DDE_Tnp_1_7 transposase domain among piggyBac proteins; however, selected members from other DNA transposon superfamilies do not show this structural feature.…”

Section: Discussionmentioning

confidence: 99%

“…It is intriguing that regardless of the acquisition of other non-transposase domains during evolution (as in the case of PGBD1 or PGBD4), the position of the disordered region is strictly kept upstream of the DDE_Tnp_1_7 transposase domain among piggyBac proteins; however, selected members from other DNA transposon superfamilies do not show this structural feature. This special structural constellation of the piggyBac -related sequences would further point to functional conservation, but the question remains: what would be the role of NTDR in PB proteins that do not show excision activities anymore [ 22 , 47 ]? A possible explanation could be that certain protein–protein interactions may well serve the new domesticated functions, and understanding the role of the NTDR in PB transposition would therefore be helpful to reveal the endogenous function of the co-opted proteins.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, the lack of potentially cross-reacting endogenous elements makes it an attractive choice for human applications [ 19 ]. Considering the PB system, there are PB-like elements in the human genome, and their cross-reactivity with the insect transposon is still a matter of debate [ 20 , 21 , 22 ]. On the other hand, the PB transposase performs a seamless excision of the transposon unit from a genomic location [ 23 , 24 , 25 ].…”

Section: Introductionmentioning

confidence: 99%

“…With a systematic bioinformatic analysis, we also show that the NTDR is specific for the piggyBac superfamily of DNA transposons and that it can be found even in domesticated, transposase-derived proteins that lost their ability to mobilize DNA. These include the five human piggyBac -derived sequences (PGBD1-5), which have been shown to have lost their mobilizing activity [ 22 ]. These results support the hypothesis that the NTDR of PB has an important regulatory function in transposition and that this regulatory function has likely been preserved during the evolution of PB-derived genes.…”

Section: Introductionmentioning

confidence: 99%

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Functional Characterization of the N-Terminal Disordered Region of the piggyBac Transposase

Wachtl

Schád

Huszár

et al. 2022

IJMS

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The piggyBac DNA transposon is an active element initially isolated from the cabbage looper moth, but members of this superfamily are also present in most eukaryotic evolutionary lineages. The functionally important regions of the transposase are well described. There is an RNase H-like fold containing the DDD motif responsible for the catalytic DNA cleavage and joining reactions and a C-terminal cysteine-rich domain important for interaction with the transposon DNA. However, the protein also contains a ~100 amino acid long N-terminal disordered region (NTDR) whose function is currently unknown. Here we show that deletion of the NTDR significantly impairs piggyBac transposition, although the extent of decrease is strongly cell-type specific. Moreover, replacing the NTDR with scrambled but similarly disordered sequences did not rescue transposase activity, indicating the importance of sequence conservation. Cell-based transposon excision and integration assays reveal that the excision step is more severely affected by NTDR deletion. Finally, bioinformatic analyses indicated that the NTDR is specific for the piggyBac superfamily and is also present in domesticated, transposase-derived proteins incapable of catalyzing transposition. Our results indicate an essential role of the NTDR in the “fine-tuning” of transposition and its significance in the functions of piggyBac-originated co-opted genes.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Functional Characterization of the N-Terminal Disordered Region of the piggyBac Transposase

Wachtl

Schád

Huszár

et al. 2022

IJMS

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Analysis of DNA transposition by DNA transposases in human cells

Bigot

Yamada

Mueller

et al. 2023

Preprint

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This manuscript discusses the recent report "Cognate restriction of transposition by piggyBac-like proteins" in Nucleic Acids Research by Beckermann et al and related recent publications about the inability to detect DNA transposition activity of human domesticated DNA transposase PGBD5. Measuring DNA transposition activity of transposases in human cells, where these enzymes can act on endogenous genomic substrates and induce DNA damage, is complicated by these and other cellular responses. Possible reasons for the discordant findings of Beckermann et al with prior independent reports of PGBD5 DNA transposition by Helou et al and Henssen et al and specific details of experimental methods in human cells are presented. In particular, by using independent experiments that reproduce PGBD5-mediated genomic integration, we demonstrate how supraphysiologic and ectopic overexpression of PGBD5 can cause DNA damage and cell death, and artifactual loss of apparent activity in clonogenic transposition reporter assays. While PGBD5 can support apparent DNA transposition, its cellular activity predominantly involves double-strand DNA breaks, deletions and other DNA rearrangements. We discuss the implications of this phenomenon for the interpretation of experimental assays and activities of domesticated DNA transposases.

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Childhood cancer mutagenesis caused by transposase-derived PGBD5

Yamada,

Keller,

Gutierrez

et al. 2024

Sci. Adv.

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Genomic rearrangements are a hallmark of most childhood tumors, including medulloblastoma, one of the most common brain tumors in children, but their causes remain largely unknown. Here, we show that PiggyBac transposable element derived 5 (Pgbd5) promotes tumor development in multiple developmentally accurate mouse models of Sonic Hedgehog (SHH) medulloblastoma. Most Pgbd5-deficient mice do not develop tumors, while maintaining normal cerebellar development. Ectopic activation of SHH signaling is sufficient to enforce cerebellar granule cell progenitor–like cell states, which exhibit Pgbd5-dependent expression of distinct DNA repair and neurodevelopmental factors. Mouse medulloblastomas expressing Pgbd5 have increased numbers of somatic structural DNA rearrangements, some of which carry PGBD5-specific sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. This identifies PGBD5 as a medulloblastoma mutator and provides a genetic mechanism for the generation of oncogenic DNA rearrangements in childhood cancer.

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Functional indications for transposase domestications – Characterization of the human piggyBac transposase derived (PGBD) activities

Cited by 5 publications

References 70 publications

Functional Characterization of the N-Terminal Disordered Region of the piggyBac Transposase

Functional Characterization of the N-Terminal Disordered Region of the piggyBac Transposase

Analysis of DNA transposition by DNA transposases in human cells

Childhood cancer mutagenesis caused by transposase-derived PGBD5

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