Reactivation of a somatic errantivirus and germline invasion in Drosophila ovaries.

Most Drosophila transposable elements are LTR retrotransposons, some of which belong to the genus Errantivirus and share structural and functional characteristics with vertebrate endogenous retroviruses. Like endogenous retroviruses, it is unclear whether errantiviruses retain some infectivity and transposition capacity. We created conditions where control of the Drosophila ZAM errantivirus through the piRNA pathway was abolished leading to its de novo reactivation in somatic gonadal cells. After reactivation, ZAM invaded the oocytes and severe fertility defects were observed. While ZAM expression persists in the somatic gonadal cells, the germline then set up its own adaptive genomic immune response by producing piRNAs against the constantly invading errantivirus, restricting invasion. Our results suggest that although errantiviruses are continuously repressed by the piRNA pathway, they may retain their ability to infect the germline and transpose, thus allowing them to efficiently invade the germline if they are expressed.

The histone demethylase Kdm3 prevents auto-immune piRNAs production in Drosophila.

Genome integrity of the animal germline is protected from transposable element activity by PIWI-interacting RNAs (piRNAs). While piRNA biogenesis is intensively explored, little is known about the genetical determination of piRNA clusters, the genomic sources of piRNAs. Using a bimodal epigenetic state piRNA cluster (BX2), we identified the histone demethylase Kdm3 as being able to prevent a cryptic piRNA production. In the absence of Kdm3, dozens of coding gene-containing regions become genuine germline dual-strand piRNA clusters. Eggs laid by Kdm3 mutant females show developmental defects phenocopying loss of function of genes embedded into the additional piRNA clusters, suggesting an inheritance of functional ovarian "auto-immune" piRNAs. Antagonizing piRNA cluster determination through chromatin modifications appears crucial to prevent auto-immune genic piRNAs production.

The catalytic-dead Pcif1 regulates gene expression and fertility in Drosophila.

Eukaryotic mRNAs are modified at the 5' end with a methylated guanosine (m7G) that is attached to the transcription start site (TSS) nucleotide. The TSS nucleotide is 2'-O-methylated (Nm) by CMTR1 in organisms ranging from insects to human. In mammals, the TSS adenosine can be further N 6 -methylated by RNA polymerase II phosphorylated CTD-interacting factor 1 (PCIF1) to create m6Am. Curiously, the fly ortholog of mammalian PCIF1 is demonstrated to be catalytic-dead, and its functions are not known. Here, we show that Pcif1 mutant flies display a reduced fertility which is particularly marked in females. Deep sequencing analysis of Pcif1 mutant ovaries revealed transcriptome changes with a notable increase in expression of genes belonging to the mitochondrial ATP synthetase complex. Furthermore, the Pcif1 protein is distributed along euchromatic regions of polytene chromosomes, and the Pcif1 mutation behaved as a modifier of position-effect-variegation (PEV) suppressing the heterochromatin-dependent silencing of the white gene. Similar or stronger changes in the transcriptome and PEV phenotype were observed in flies that expressed a cytosolic version of Pcif1. These results point to a nuclear cotranscriptional gene regulatory role for the catalytic-dead fly Pcif1 that is probably based on its conserved ability to interact with the RNA polymerase II carboxy-terminal domain.

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 (see decision letter).

Transcriptional profiling of Klebsiella pneumoniae defines signatures for planktonic, sessile and biofilm-dispersed cells.

BACKGROUND: Surface-associated communities of bacteria, known as biofilms, play a critical role in the persistence and dissemination of bacteria in various environments. Biofilm development is a sequential dynamic process from an initial bacterial adhesion to a three-dimensional structure formation, and a subsequent bacterial dispersion. Transitions between these different modes of growth are governed by complex and partially known molecular pathways. RESULTS: Using RNA-seq technology, our work provided an exhaustive overview of the transcriptomic behavior of the opportunistic pathogen Klebsiella pneumoniae derived from free-living, biofilm and biofilm-dispersed states. For each of these conditions, the combined use of Z-scores and principal component analysis provided a clear illustration of distinct expression profiles. In particular, biofilm-dispersed cells appeared as a unique stage in the bacteria lifecycle, different from both planktonic and sessile states. The K-means cluster analysis showed clusters of Coding DNA Sequences (CDS) and non-coding RNA (ncRNA) genes differentially transcribed between conditions. Most of them included dominant functional classes, emphasizing the transcriptional changes occurring in the course of K. pneumoniae lifestyle transitions. Furthermore, analysis of the whole transcriptome allowed the selection of an overall of 40 transcriptional signature genes for the five bacterial physiological states. CONCLUSIONS: This transcriptional study provides additional clues to understand the key molecular mechanisms involved in the transition between biofilm and the free-living lifestyles, which represents an important challenge to control both beneficial and harmful biofilm. Moreover, this exhaustive study identified physiological state specific transcriptomic reference dataset useful for the research community.

Spatio-temporal requirements for transposable element piRNA-mediated silencing during Drosophila oogenesis.

During Drosophila oogenesis, transposable element (TE) repression involves the Piwi-interacting RNA (piRNA) pathway which ensures genome integrity for the next generation. We developed a transgenic model to study repression of the Idefix retrotransposon in the germline. Using a candidate gene KD-approach, we identified differences in the spatio-temporal requirements of the piRNA pathway components for piRNA-mediated silencing. Some of them (Aub, Vasa, Spn-E) are necessary in very early stages of oogenesis within the germarium and appear to be less important for efficient TE silencing thereafter. Others (Piwi, Ago3, Mael) are required at all stages of oogenesis. Moreover, during early oogenesis, in the dividing cysts within the germarium, Idefix anti-sense transgenes escape host control, and this is associated with very low piwi expression. Silencing of P-element-based transgenes is also strongly weakened in these cysts. This region, termed the 'Piwiless pocket' or Pilp, may ensure that new TE insertions occur and are transmitted to the next generation, thereby contributing to genome dynamics. In contrast, piRNA-mediated silencing is strong in germline stem cells in which TE mobilization is tightly repressed ensuring the continued production of viable germline cysts.

Drosophila heterochromatin: structure and function.

Heterochromatic domains, which are enriched in repetitive sequences and packaged in a higher-order chromatin folding, carry the potential to epigenetically inactivate a euchromatic gene that has been moved in close proximity. The discovery that these domains encode non-coding RNAs involved in RNA-silencing mechanisms has recently contributed to a better understanding of the mechanisms of the epigenetic repression established by heterochromatic domains. In this review, we will consider the repeated nature of their DNA sequence, the successive steps in heterochromatin assembly, starting with the decision process, the higher order state assembly and its epigenetic propagation. Recent findings provide new insights into the cellular functions of heterochromatin, notably its major contribution to genome stability and chromosome integrity.