Mechanisms of human LINE-1 retrotransposition and DNA repair
Zusammenfassung der Projektergebnisse
By analyzing 5’ junctions of human LINE-1 and Alu retrotransposons, we were able to shed light on the mechanism of the attachment of the 5’ end of newly synthesized L1 cDNAs to the genomic target DNA during L1 retrotransposition. Based on the microhomologies at the 5’ junctions of 5’-truncated standard L1 insertions, we propose a model for the generation of 5’- truncated L1 integrants in which joining of the cDNA to the genomic target DNA and subsequent second-strand synthesis are carried out by host-encoded factors that are involved in double-strand break repair by ‘error-prone’ non-homologous end joining (NHEJ). In order to identify those host-encoded DSB repair factors and/or cell cycle monitoring proteins involved in L1 retrotransposition, we knocked down several of these factors and evaluated the consequences for retrotransposition of a marked L1 reporter element. We found that p53 knockdown reduces retrotransposition of the L1 reporter element in HeLa cells by ~30 %. This result is consistent with a 3-to-4-fold increase of the L1 retrotransposition rate after overexpression of wildtype p53 in H1299 cells. Since wt p53 was shown to bind to the L1 promoter region, p53 seems to act as transcription factor and regulate L1 retrotransposition rate by transcriptional activation. Analysis of the impact of other factors involved in DSB repair and cell cycle monitoring on L1 retrotransposition is still ongoing. In order to allow for concurrent L1 reporter expression and DSB repair factor knock down in our transient cotransfection experiments, we subjected the L1 retrotransposition reporter cassette to the tetracyclin-controlled gene expression system. We established a doxycyclin-inducible system for conditional activation of human L1 reporter elements in a time- and dose-dependent manner. This inducible retrotranspostion system has industrial applications: i) The invention facilitates random insertional mutagenesis in cells and transgenic organisms in defined tissues and during well defined and controlled periods of time. The tightly controlled activation and deactivation of L1 retrotransposition at defined time points facilitates the identification of gene functions in general and at defined developmental stages. Conditional activation of the L1 retrotransposition in defined somatic tissues will help to identify genes involved in cancer development. The relevance for the pharmaceutical industry is obvious. ii) Ex vivo gene therapy approaches with L1/Adenovirus– hybrid vectors or with other L1-based gene delivery systems: If the therapeutic gene is part of the inducible L1, controlling the time point of L1 retrotransposition will define the start of therapeutic gene expression. We identified and isolated the first functional rat L1 retrotransposon and demonstrated that functional rat L1 elements are expressed and mobilized in rat chloroleukemia (RCL) cells. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia. It was also demonstrated that downregulation of endogenous L1 expression in human tumor cell lines reduced proliferation, induced morphological differentiation and reprogrammed gene expression. Inhibition of L1 expression antagonized tumor growth in mice. LINE1–encoded endonuclease (L1-EN) bears considerable technological interest, because its target selectivity may ultimately be engineered to allow the site-specific integration of DNA into defined genomic locations. By generating loop grafts of L1-EN, we demonstrate that they result in altered specificity, while individual point mutations do not change the nicking pattern of L1-EN. We show that structural parameters of the DNA targets are more important for recognition than the nucleotide sequence, and nicking profiles on DNA oligonucleotides in vitro are less well defined than the respective integration site consensus in vivo. This suggests that additional factors other than the DNA nicking specificity of L1-EN contribute to the targeted integration of non-LTR retrotransposons.
Projektbezogene Publikationen (Auswahl)
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(2005). Analysis of 5’ junctions of human LINE-1 and Alu retrotransposons suggests an alternative model for 5’ end attachment requiring nonhomologous end joining. Genome Res. 15: 780-789
Zingler, N., Willhoeft, U., Brose, H.-P., Schoder, V., Jahns, T., Hanschmann, K.-M. O., Morrish, T.A., Löwer, J., and Schumann, G.G.
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(2007). Determinants for DNA target structure selectivity of the human LINE-1 retrotransposon endonuclease. Nucleic Acids Res. 35: 4914-26
Repanas, K., Zingler N., Layer, L.E., Schumann, G.G., Perrakis, A., and Weichenrieder, O.
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(2007). Distinct roles for LINE-1 and HERV-K retroelements in cell proliferation, differentiation and tumor progression. Oncogene 26: 4226-33
Oricchio E., Sciamanna, I., Beraldi, R., Tolstonog, G.V., Schumann, G.G., and Spadafora, C.
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(2008). Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells. Nucleic Acids Res. 36: 646-665
Kirilyuk, A., Tolstonog, G.V., Damert, A., Held, U., Hahn, S., Löwer, R., Buschmann, C., Horn, A.V., Traub, P., and Schumann, G.G.
