What is the problem/issue being addressed?
The main goal of the proposed project was to analyse the epigenetic mechanisms involved in germ cell identity and maintenance. The Polycomb Group (PcG) protein machinery acts in the nucleus to epigenetically silence genes and seems to be critical for germ cell development. The Piwi machinery acts in the cytoplasm to regulate gene expression at the post transcriptional level, which has been shown to be essential for germ cell maintenance. We wanted to know if and how PcG and Piwi machineries act together during early development and germ cell specification/maintenance.
Why is it important for society?
Because of a fundamental character of our research, it is hard to disseminate the direct impact of the results we obtained in our studies on society. We believe that our results, besides being an important resource for functional PcG gene studies in-vivo, constitute a major advance to further our understanding of the mechanisms of epigenetic, and more specific PcG protein-mediated, gene regulation during vertebrate germ line development and embryogenesis. The extensive evidence linking PcG protein, Ezh2 activity to cancer has prompted interest in the underlying mechanism. Therefore we focused our research on understanding the role of Ezh2 in early development and germ cell specification, in order to hopefully disseminate our results on more effective clinical applications in the future.
What are the overall objectives?
The project had two main experimental objectives :
1. Mapping epigenetic marks during development in vertebrates.
2. Identification of the role of nuclear complexes (PRC1, PRC2) and germ granule proteins (Piwi proteins Ziwi/Zili) and their possible cross-talk in maintaining germ cell identity by epigenetic gene regulation in the nucleus.
Conclusions of the action:
We have investigated in great detail the gene expression profile of PcG genes during zebrafish embryonic and germ line development and published it in PLOS ONE (open access). In adults, expression of selected PcG genes is found to be enriched in germ line over somatic tissues (Figure attached). Therefore, the germ line of adult zebrafish was analysed for the expression pattern of a selection of PcG genes by whole mount in-situ hybridization. We detected presence of the tested PcG gene transcripts at early stages of both oogenesis and spermatogenesis. This enriched expression for early stages of gametogenesis is also observed in developing gonads at 4 and 5 weeks post fertilization. Additionally, zebrafish embryos were used to study the spatio-temporal expression patterns of a selection of PcG genes during embryonic development. The PcG genes that we tested are maternally loaded and ubiquitously expressed at early developmental stages, except of ezh1. The expression of the PcG genes that were assessed became enriched anteriorly over time, and is more spatially defined during tissue specification. The data shown here is an important resource for functional PcG gene studies in-vivo.
We have also analysed the loss of the PcG component Ezh2 during zebrafish embryogenesis – work that we have submitted to be published in eLife. We found that zebrafish body plan formation is independent of PcG-mediated gene repression, despite impaired epigenetic control, until the stage of tissue maintenance where it becomes required to maintain spatially restricted transcriptional profiles of transcription factors. We also investigated the effect of the loss of Ezh2 on germline development by performing RNA-seq analysis on ovaries and testis lacking both maternal and zygotic Ezh2 (material possible to be obtained only by unique germ cell transplantation method optimised in our group). We are currently still analysing this unique and complete dataset and hope to publish it shortly.
Despite enormous effort and time invested, we were unable to confirm our hypothesis that the PIWI machinery contributes to or shows cross-talk with epigenetic control of the genome, by direct or indirect interaction with PcG proteins (like Ezh2). However, we confirmed that shortly after insertion of a transposon in the genome, they are silenced by newly produced piRNAs and the older the inserted transposons, the less likely they are targeted by PIWI machinery.
Figure legend: Expression of the majority of the PcG genes that were tested is enriched in the adult germ line.(A) Relative expression assessed by RT-qPCR. Error bars indicate standard deviation. (B) Spatio-temporal expression of genes in adult ovaries. Scale bar: 100 μm. (C) Expression of the PcG genes in different stages of oogenesis. (D) Spatio-temporal expression of ezh2, eed, suz12a, phc2a, and bmi1a in adult testes. Scale bar: 100 μm. (E) Expression of the PcG genes in different stages of spermatogenesis.
