Epigenetic silencing by small ovarian RNAs – ESSOR

Our project is based on genetic crosses and on the high-throughput sequencing of small non coding RNAs. During the first 6 month, we have acquired the expertise in small non coding RNA extraction from drosophila ovaries and embryos. We have also learned how to analyze the deep sequencing data set and now we are independent for the analysis of this type of data.

All the tasks of our project have already started and in a time window allowing to expect that the project will be delivered in time. Moreover, one of the task on the role of piRNAs in the transmission of an acquired trait has already given interesting results which have been published in a scientific revue called Genome Research (Impact factor 13,5). This work demonstrates that small non coding RNAs can be transmitted from the mother to the daughter and can influence the transposable elements regulation in the progeny. In other words, the epigenetic marks are not only present on the chromatin but can also be transmitted through generations via the small non coding RNA populations.

Based on the fundamental data obtained in plants and yeast, the role played by small non coding RNA seems to be underestimated in the pluricellular eukaryotes. We would like to demonstrate that the piRNAs play a crucial role in the maintenance of genome integrity. Studying their biogenesis and their role could allow to understand why in some cancers like colon, cervical and gastric cancers the proteins that load the small non coding RNAs are reexpressed like in the populations of undifferentiated cells.
Our work opens new perspectives in the epigenetic area because it shows that the epigenetic information is not only explained by chromatin marks but also by small non coding RNAs.

Grentzinger T, Armenise C, Brun C, Mugat B, Serrano V, Pelisson P, Chambeyron S. (2012)
piRNA-mediated transgenerational inheritance of an acquired trait. Genome Research May 3. [Epub ahead of print].

The aim of this project is to elucidate the mechanisms involved in the control of transposable elements (TEs). These mechanisms are essential for the maintenance of genome integrity. They involve a class of small RNAs, the piRNAs (piwi-interacting RNAs). The piRNA-associated silencing pathway is not well known. We propose to characterize the essential steps of this pathway in the Drosophila ovary.
piRNAs may be considered as the key elements of a sort of bipartite immune system: one genetic component is encoded by heterochromatic loci that contain defective copies of TEs (piRNA clusters) producing antisense piRNAs; the other component is adaptive and corresponds to the sense piRNAs produced by the functional copies of TEs located in euchromatin. The proposed repression model associated with piRNAs is as follows: primary antisense piRNAs, produced by an unknown mechanism from piRNA clusters, target the transcripts of functional TEs which are cut to produce sense piRNAs. The latter target transcripts of the piRNA clusters that are cut in turn to produce secondary antisense piRNAs.
Our recent results show that such an amplification loop very likely occurs in the female germ line but does not occur in the ovarian somatic cells where only the primary piRNAs are present.
Based on our knowledge on two TEs , the I element in the germ line and gypsy in the soma, we will study the biogenesis of the primary piRNAs, the role of piRNAs in TE repression, and the epigenetic mechanisms involved in the maternal inheritance of this silencing.
First, we will focus on the biogenesis of the primary piRNAs in the ovarian somatic tissue (that is devoid of secondary piRNAs), by testing the hypothesis that they are cleavage products of long heterochromatic transcripts containing the antisense strand of TEs. We will determine if Drosha, a RNAse III involved in the production of miRNAs, is involved in the processing of these transcripts in piRNAs, using a dominant negative mutant that we already have got. We will also test the putative role of TE siRNAs in the processing of the piRNA precursors. Conversely, we will ask if piRNAs can repress the biosynthesis of siRNAs by reducing the amounts of TE double stranded RNAs. If it is the case, it will be a strong evidence supporting the idea of the occurrence of a regulation feed-back between siRNAs and piRNAs allowing an optimal repression of TEs.
The repression level of the I element in the female germ line depends on the age and on various environmental conditions such as temperature. Its variations are maternally transmitted through generations. Hence, this element offers the opportunity to study epigenetic features that are maternally transmitted over many generations. The molecular mechanisms of this transgenerational transmission are still unknown and piRNAs (some of which are maternally deposited in the embryo) could be the maternally transmitted epigenetic factors. We will characterize quantitatively and qualitatively the piRNA populations maternally deposited in the embryos. This part of the project will involve deep sequencing of the small RNAs present in the ovaries and in the embryos of isogenic strains differing by the capacity to repress the I element. We will then ask if these strains differ also by epigenetic marks on the chromatin associated to piRNA clusters.
In order to determine the role of the piRNAs maternally deposited, we will try to modify the repression level of I elements by microinjecting embryos with piRNAs identified by the deep sequencing analyses. If an effect is observed, we will ask if it is also associated to chromatin modifications.
This project will help to understand the mechanisms involved in the maintenance of genome integrity determined by piRNAs and the nature of the epigenetic modifications transmitted through maternal generations.