Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway.

Germline genes often become re-expressed in soma-derived human cancers as "cancer/testis antigens" (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. We found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer.We thank the Cold Spring Harbor Laboratory Microscopy Shared Resources for assistance, which are funded in part by Cancer Center Support Grant 5P30CA045508. This work was supported in part by a grant from the STARR Cancer Consortium, grants from the National Institutes of Health (NIH MERIT Award, R37GM062534 to G. J.H.), and a generous gift from Kathryn W. Davis to G.J.H. N.P. and G.J.H. are or were Investigators of the Howard Hughes Medical Institute. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. Cell lines have been deposited by A.S. at the Drosophila Genomics Resource Center (NIH 2P40OD010949-10A1). G.J.H. is supported by Cancer Research UK and is a Wellcome Trust Investigator.This is the final version of the article. It first appeared from Cold Spring Harbor Press at http://dx.doi.org/10.1101/gad.284927.116

Loss of the Tumor Suppressor Pten Promotes Proliferation of Drosophila melanogaster Cells In Vitro and Gives Rise to Continuous Cell Lines

In vivo analysis of Drosophila melanogaster has enhanced our understanding of many biological processes, notably the mechanisms of heredity and development. While in vivo analysis of mutants has been a strength of the field, analyzing fly cells in culture is valuable for cell biological, biochemical and whole genome approaches in which large numbers of homogeneous cells are required. An efficient genetic method to derive Drosophila cell lines using expression of an oncogenic form of Ras (RasV12) has been developed. Mutations in tumor suppressors, which are known to cause cell hyperproliferation in vivo, could provide another method for generating Drosophila cell lines. Here we screened Drosophila tumor suppressor mutations to test if they promoted cell proliferation in vitro. We generated primary cultures and determined when patches of proliferating cells first emerged. These cells emerged on average at 37 days in wild-type cultures. Using this assay we found that a Pten mutation had a strong effect. Patches of proliferating cells appeared on average at 11 days and the cultures became confluent in about 3 weeks, which is similar to the timeframe for cultures expressing RasV12. Three Pten mutant cell lines were generated and these have now been cultured for between 250 and 630 cell doublings suggesting the life of the mutant cells is likely to be indefinite. We conclude that the use of Pten mutants is a powerful means to derive new Drosophila cell lines

Efficient Genetic Method for Establishing Drosophila Cell Lines Unlocks the Potential to Create Lines of Specific Genotypes

Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene RasV12 (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of RasV12 is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype

Genome-wide activities of Polycomb complexes control pervasive transcription

Polycomb group (PcG) complexes PRC1 and PRC2 are well known for silencing specific developmental genes. PRC2 is a methyltransferase targeting histone H3K27 and producing H3K27me3, essential for stable silencing. Less well known but quantitatively much more important is the genome-wide role of PRC2 that dimethylates similar to 70% of total H3K27. We show that H3K27me2 occurs in inverse proportion to transcriptional activity in most non-PcG target genes and intergenic regions and is governed by opposing roaming activities of PRC2 and complexes containing the H3K27 demethylase UTX. Surprisingly, loss of H3K27me2 results in global transcriptional derepression proportionally greatest in silent or weakly transcribed intergenic and genic regions and accompanied by an increase of H3K27ac and H3K4me1. H3K27me2 therefore sets a threshold that prevents random, unscheduled transcription all over the genome and even limits the activity of highly transcribed genes. PRC1-type complexes also have global roles. Unexpectedly, we find a pervasive distribution of histone H2A ubiquitylated at lysine 118 (H2AK118ub) outside of canonical PcG target regions, dependent on the RING/Sce subunit of PRC1-type complexes. We show, however, that H2AK118ub does not mediate the global PRC2 activity or the global repression and is predominantly produced by a new complex involving L(3) 73Ah, a homolog of mammalian PCGF3

Transcriptional Control of an Essential Ribozyme in <i>Drosophila</i> Reveals an Ancient Evolutionary Divide in Animals

<div><p>Ribonuclease P (RNase P) is an essential enzyme required for 5′-maturation of tRNA. While an RNA-free, protein-based form of RNase P exists in eukaryotes, the ribonucleoprotein (RNP) form is found in all domains of life. The catalytic component of the RNP is an RNA known as RNase P RNA (RPR). Eukaryotic <i>RPR</i> genes are typically transcribed by RNA polymerase III (pol III). Here we showed that the <i>RPR</i> gene in <i>Drosophila</i>, which is annotated in the intron of a pol II-transcribed protein-coding gene, lacks signals for transcription by pol III. Using reporter gene constructs that include the RPR-coding intron from <i>Drosophila</i>, we found that the intron contains all the sequences necessary for production of mature RPR but is dependent on the promoter of the recipient gene for expression. We also demonstrated that the intron-coded RPR copurifies with RNase P and is required for its activity. Analysis of <i>RPR</i> genes in various animal genomes revealed a striking divide in the animal kingdom that separates insects and crustaceans into a single group in which <i>RPR</i> genes lack signals for independent transcription and are embedded in different protein-coding genes. Our findings provide evidence for a genetic event that occurred approximately 500 million years ago in the arthropod lineage, which switched the control of the transcription of <i>RPR</i> from pol III to pol II.</p></div

<i>Drosophila RPR</i> is embedded in an intron and ubiquitously expressed.

<p>A. The <i>RPR</i> gene (pink) in <i>D. melanogaster</i> is present in the last intron of <i>ATPsynC</i>/<i>CG1746</i>. B. This arrangement is conserved in <i>D. pseudoobscura</i> (and other members of the genus<i>).</i> The exons of <i>ATPsynC</i> (orange peaks) are highly conserved between these species, as is the region within the intron that contains <i>RPR</i> (pink). The preceding intron is not conserved. Untranslated regions of <i>ATPsynC</i> are shown in grey. Peaks showing 75% or greater conservation are colored. C. Analysis of polyA-selected RNA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-Graveley1" target="_blank">[52]</a> from <i>D. pseudoobscura</i> and <i>D. virilis,</i> and of total RNA from different developmental stages of <i>D. melanogaster</i> show that the region corresponding to <i>RPR</i> is expressed at higher levels than the preceding intron <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-Graveley1" target="_blank">[52]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-Roy1" target="_blank">[53]</a>. Presence of RPR in polyA⁺ RNA is likely due to carryover (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#s4" target="_blank">Materials and Methods</a>). D. ChIP on chip data (<i>D. melanogaster</i> embryos) showing binding sites of pol II <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-Li2" target="_blank">[63]</a> and transcription factor IIB (TF-IIB) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004893#pgen.1004893-MacArthur1" target="_blank">[64]</a> in the 5′ region of <i>ATPsynC/CG1746</i>. E, embryonic stage in hours after egg laying; L, larval instar; WPP, white pre-pupae; F, female; M, male.</p