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Cell ; 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38843831


Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

Cell ; 186(13): 2865-2879.e20, 2023 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-37301196


Retroelements are the widespread jumping elements considered as major drivers for genome evolution, which can also be repurposed as gene-editing tools. Here, we determine the cryo-EM structures of eukaryotic R2 retrotransposon with ribosomal DNA target and regulatory RNAs. Combined with biochemical and sequencing analysis, we reveal two essential DNA regions, Drr and Dcr, required for recognition and cleavage. The association of 3' regulatory RNA with R2 protein accelerates the first-strand cleavage, blocks the second-strand cleavage, and initiates the reverse transcription starting from the 3'-tail. Removing 3' regulatory RNA by reverse transcription allows the association of 5' regulatory RNA and initiates the second-strand cleavage. Taken together, our work explains the DNA recognition and RNA supervised sequential retrotransposition mechanisms by R2 machinery, providing insights into the retrotransposon and application reprogramming.

RNA , Retroelementos , RNA/metabolismo , Clivagem do DNA , DNA Polimerase Dirigida por RNA/metabolismo , Transcrição Reversa
Cryobiology ; 90: 41-46, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31513810


Oocyte vitrification has extensively been applied in the field of embryo engineering and in the preservation of genetic resources of fine livestock. Following our previous work in oocyte vitrification and the level change of DNA methylation, here we further explored the dynamic change of three active demethylation proteins: Ten-Eleven-Translocation 1/2/3(TET1/2/3), 5-methylcytosine (5 mC) and 5-hydroxymethycytosine (5hmC) after vitrification and warming. In order to observe the active demethylation in vitrified oocytes, two small molecular regulators, i.e. Vitamin C (VC) and dimethyloxaloylglycine (DMOG) were used to adjust activity and level of the TET 3 protein. The results showed that the levels of 5 mC and 5hmC were significantly decreased after 2 h of vitrification (P < 0.01). Moreover, the level of TET3 protein was significantly increased after 2 h warming (P < 0.01). And the relative gene expression of TET2/3 did not change in the first 2 h, but significantly increased after 2 h (P < 0.01). When VC was added to vitrification and recovery medium, it could not significantly improve the level of TET3 gene expression, and affect 5 mC and 5hmC expression (P > 0.05). When the DMOG was added to the solutions of vitrification, the level of 5hmC showed significantly increase (P < 0.01). In conclusion, the oocyte vitrification procedure reduced DNA methylation and hydroxymethylation in MII oocytes, but adding VC and DMOG to vitrification medium can prevent the reduction of DNA hydroxymethylation by increasing activity of TET3 methylation protein after vitrification and warming.

Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Oócitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Vitrificação , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Aminoácidos Dicarboxílicos/farmacologia , Animais , Ácido Ascórbico/farmacologia , Criopreservação/métodos , Crioprotetores/farmacologia , Pesquisas com Embriões , Feminino , Expressão Gênica , Camundongos
Mol Reprod Dev ; 86(7): 862-870, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31066155


Resveratrol (Res) has been reported to be able to improve oocyte vitrification because of its antioxidative properties. The objective of this study was to further assess the positive effect of Res addition on the developmental potential of vitrified mouse oocytes from the perspective of epigenetic alterations. First, 2 µM Res was chosen as the optimal concentration on the basis of its effects on survival and its antioxidative properties. We found that Res addition significantly promoted fertilization (63.8% vs. 42.9%) and blastocyst formation (68.3% vs. 50.2%) after oocyte vitrification. The quality of the derived blastocysts was also higher after Res treatment. Regarding epigenetic aspects, the expression of the important deacetylase SIRT1 was found to decrease significantly upon vitrification, but it was rescued by Res. The abnormal levels of H3K9 acetylation and DNA methylation in vitrified oocytes were restored by Res addition. Moreover, the expression of several imprinted genes was affected by oocyte vitrification. Among them, abnormal Gtl2 and Peg3 expression levels were restored by Res addition. Therefore, the methylation of their imprinted control regions (ICRs) was examined. Surprisingly, the abnormal patterns of Gtl2 and Peg3 methylation in blastocysts developed from vitrified oocytes were both restored by Res addition. Finally, the full-term embryonic development showed that the birth rate was improved significantly by Res addition (56.2% vs. 38.1%). Collectively, Res was beneficial for the pre- and postimplantation embryonic development. Except for the antioxidative activity, Res also played a role in the correction of some abnormal epigenetic modifications caused by oocyte vitrification.

Antioxidantes/farmacologia , Desenvolvimento Embrionário/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Resveratrol/farmacologia , Vitrificação , Animais , Blastocisto/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Transferência Embrionária , Feminino , Fertilização in vitro/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Masculino , Camundongos , Oócitos/metabolismo , Gravidez , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo