Untersuchung des Mechanismus der piRNA vermittelten epigenetischen Stilllegung von Transposons während der fötalen Keimzell-Reprogrammierung
Allgemeine Genetik und funktionelle Genomforschung
Zellbiologie
Zusammenfassung der Projektergebnisse
The funded project resulted in the characterisation of the MIWI2 interactome in vivo in mouse foetal germ cells, which led to the break-through discovery of the uncharacterized protein SPOCD1 as an essential executor of MIWI2-directed transposon DNA methylation and silencing (Zoch et al., 2020). Similar to MIWI2, expression of the MIWI2-interacting protein SPOCD1 is restricted to male foetal germ cells undergoing de novo DNA methylation and it contains a nuclear localisation signal, which hinted at a potential role downstream of MIWI2. Analysis of mice harbouring a Spocd1null allele revealed that loss of Spocd1 strikingly resembles the phenotype of loss of Miwi2 in regard to fertility, expression of specific transposon classes and loss of genomic DNA methylation specifically on evolutionary young, full-length transposable elements. Importantly, loss of Spocd1 did not interfere with piRNA biogenesis or de novo DNA methylation, which placed SPOCD1 downstream of MIWI2 in the piRNA pathway. Through generation and analysis of an HA epitope-tagged Spocd1 allele we found that SPOCD1 interacts with several chromatin-associated proteins, notably several members of the NURD and BAF chromatin remodelling complexes and the de novo DNA methylation proteins DNMT3A and DNMT3L. Thus, this study described the first nuclear mediator of MIWI2’s epigenetic silencing function, associated for the first time chromatin remodelling activity with the mammalian piRNA pathway and importantly, provided rich datasets of MIWI2 and SPOCD1 interacting proteins that have opened the field and allow the study of many more factors potentially required for piRNA-mediated transposon silencing. From these datasets we identified a second nuclear factor, TEX15, as an essential executor of MIWI2-directed transposon DNA methylation. Notably, in contrast to Miwi2 and Spocd1, expression of Tex15, although specific to male germ cells, extends beyond the phase of de novo DNA methylation into adult spermatogenesis. Moreover, while the loss of Tex15 phenotype was similar to the loss of Spocd1 or Miwi2 phenotypes, TEX15 was only identified as an interactor of MIWI2, not of SPOCD1. TEX15 might therefore be placed upstream of SPOCD1 or alternatively be engaged by MIWI2 in parallel to SPOCD1 for the silencing of transposons and recruitment of the de novo DNA methylation machinery. The identification of SPOCD1 and TEX15 as nuclear effectors of the piRNA pathway as well as the generation of MIWI2 and SPOCD1 in vivo interactome datasets present a major advancement for the field, commented on in Nature Structural & Molecular Biology. These achievements have pushed through the scientific bottleneck that was the reliance on sparse in vivo material from foetal germ cells for the study of piRNA pathway-mediated epigenetic transposon silencing and now allows the study of nuclear effectors of the piRNA pathway in more easily accessible systems, for example through expression in cell lines or biochemical analysis of recombinant proteins.
Projektbezogene Publikationen (Auswahl)
- 2019. "PIWI- interacting RNAs: small RNAs with big functions." Nat Rev Genet 20 (2):89-108
Ozata, D. M., Gainetdinov, I., A. Zoch, D. O'Carroll, and P. D. Zamore
(Siehe online unter https://doi.org/10.1038/s41576-018-0073-3) - 2020. "SPOCD1 is an essential executor of piRNA-directed de novo DNA methylation." Nature 584 (7822):635-639
Zoch, A., T. Auchynnikava, R. V. Berrens, Y. Kabayama, T. Schöpp, M. Heep, L. Vasiliauskaitė, Y. A. Pérez-Rico, A. G. Cook, A. Shkumatava, J. Rappsilber, R. C. Allshire, and D. O'Carroll
(Siehe online unter https://doi.org/10.1038/s41586-020-2557-5) - 2020. "TEX15 is an essential executor of MIWI2-directed transposon DNA methylation and silencing." Nat Commun 11 (1):3739
Schöpp, T., A. Zoch, R. V. Berrens, T. Auchynnikava, Y. Kabayama, L. Vasiliauskaitė, J. Rappsilber, R. C. Allshire, and D. O'Carroll
(Siehe online unter https://doi.org/10.1038/s41467-020-17372-5)
