Generation of Trophoblast-Like Cells From Hypomethylated Porcine Adult Dermal Fibroblasts

The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is the progenitor of the outer epithelial components of the placenta, and which supports the fetus during the intrauterine life. However, the epigenetic and paracrine controls at work in trophectoderm differentiation are still to be fully elucidated and the creation of dedicated in vitro models is desirable to increase our understanding. Here we propose a novel approach based on the epigenetic conversion of adult dermal fibroblasts into trophoblast-like cells. The method combines the use of epigenetic erasing with an ad hoc differentiation protocol. Dermal fibroblasts are erased with 5-azacytidine (5-aza-CR) that confers cells a transient high plasticity state. They are then readdressed toward the trophoblast (TR) phenotype, using MEF conditioned medium, supplemented with bone morphogenetic protein 4 (BMP4) and inhibitors of the Activin/Nodal and FGF2 signaling pathways in low O2 conditions. The method here described allows the generation of TR-like cells from easily accessible material, such as dermal fibroblasts, that are very simply propagated in vitro. Furthermore, the strategy proposed is free of genetic modifications that make cells prone to instability and transformation. The TR model obtained may also find useful application in order to better characterize embryo implantation mechanisms and developmental disorders based on TR defects.

Environmentally-induced epigenetic conversion of a piRNA cluster

Transposable element (TE) activity is repressed in animal gonads by PIWI-interacting RNAs (piRNAs) produced by piRNA clusters. Current models in flies propose that germinal piRNA clusters are functionally defined by the maternal inheritance of piRNAs produced during the previous generation. Taking advantage of an inactive, but ready to go, cluster of P-element derived transgene insertions in Drosophila melanogaster, we show here that raising flies at high temperature (29°C) instead of 25°C triggers the stable conversion of this locus from inactive into actively producing functional piRNAs. The increase of antisense transcripts from the cluster at 29°C combined with the requirement of transcription of euchromatic homologous sequences, suggests a role of double stranded RNA in the production of de novo piRNAs. This report describes the first case of the establishment of an active piRNA cluster by environmental changes in the absence of maternal inheritance of homologous piRNAs.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

Environmentally-induced epigenetic conversion of a piRNA cluster

ABSTRACTTransposable element (TE) activity is repressed in animal gonads by PIWI-interacting RNAs (piRNAs), a class of small RNAs produced by specific loci made of TEs insertions and fragments. Current models propose that these loci are functionally defined by the maternal inheritance of piRNAs produced during the previous generation, raising the question of their first activation in the absence of piRNAs. Taking advantage of an inactive cluster of P-element derived transgene insertions, we show here that raising flies at high temperature (29°C) instead of 25°C results in a rare but invasive epigenetic conversion of this locus into an active piRNAs producing one. The newly acquired epigenetic state is stable over many generations even when flies are switch back to 25°C. The silencing capacities, piRNA production and chromatin modifications of the cluster are all identical whether conversion occurred by maternal piRNA inheritance or by high temperature. We also demonstrate that in addition to high temperature, a single homologous transgene inserted elsewhere in the genome is required to activate the locus. We thus have identified a minimal system of three components to create a stable piRNA producing locus: 1) a locus with multiple TE derived sequences; 2) an euchromatic copy of these sequences and 3) elevated temperature. Altogether, these data report the first case of the establishment of an active piRNA cluster by environmental changes. It highlights how such variations of species natural habitat can become heritable and shape their epigenome.SIGNIFICANCE STATEMENTRecently, we have witnessed great progress in our understanding of the silencing of Transposable Elements (TEs) by piRNAs, a class of small RNAs produced by piRNA clusters. At each generation, piRNA clusters are supposed to be activated by homologous piRNAs inherited from the mother raising the question of the making of the first piRNAs. Here, we report the birth of a stable and functional piRNA cluster induced by high temperature without maternal inheritance of homologous piRNAs. We propose a minimal system to create a piRNA cluster: a sufficient number of repeated sequences, a euchromatic copy of these sequences and an increase in the production of antisense RNA.