Project description:The PiwiM-bM-^@M-^SpiRNA complex (PiwiM-bM-^@M-^SpiRISC) in Drosophila ovarian somatic cells represses transposons transcriptionally to maintain genome integrity; however, the underlying mechanisms remain obscure. We performed mRNA-seq analysis from OSCs transfected with siRNAs against CG3893, the known piRNA pathway genes, Piwi, Maelstrom, HP1a and Armitage, and the control (EGFP), and PolII ChIP-seqanalysis from OSCs transfected with siRNAs against CG3893, Piwi, Mael and the control (EGFP). This result indicates that CG3893 is a novel factor for primary piRNA pathway in OSCs. RNA levels in wild-type (EGFP control knock-down) ovarian somatic cells (OSC) and RNAi knock-downs of Piwi, Armi, Mael, CG3893, and HP1a. RNA Polymerase II occupancy in wild-type (EGFP control knock-down) ovarian somatic cells (OSC) and RNAi knock-downs of Piwi, Mael, and CG3893.
Project description:Cardiac remodelling is a key process in the development of heart failure. Reactivation of foetal cardiac genes is often associated with cardiac remodelling, which might be the result of the activation of signalling pathways involved in heart failure. Here we studied the role of Pontin (Ruvbl1), which is highly expressed in embryonic hearts, in mediating adverse remodelling in adult mouse hearts.
Project description:Somatic STAT5B gain-of-function mutations have been frequently found in patients with T- and NK-cell neoplasms. STAT5BN642H represents the most frequently occuring STAT5B mutation. To investigate the molecular mechanism of STAT5BN642H-driven NK-cell leukemia, we performed RNA-Seq of liver derived FACS-sorted diseased N642HNK/NK and aged non-diseased control (Cre neg, GFPNK/NK), STAT5BNK/NK, N642HNK/NK NK cells.
Project description:This experiment was perfomed to understand the role of Cyclin C in BCR-ABL1 driven leukemia. Bone marrow from mice harbouring a homozygous Cyclin C knock-out as well as from wild type control mice was transduced with a BCR-ABL1 expressing retrovirus to generate stable cell lines. Cells were harvested for RNA seq from in-vitro cultures of these cell lines (\\"in-vitro\\" samples). 500000 cells of the same cultures were injected into the tail vains of NSG mice. After 10 days the cells were isolated from the bone marrow of the recipient mice and collected for RNA sequencing (\\"ex-vivo\\" samples). Library prep and sequencing was perfomed for in-vitro and ex-vivo samples together.
Project description:Background: Transposable elements (TEs) represent a substantial fraction of the genomes, playing a major role in evolution, as sources of genetic variability. To fully appraise the role of TE in the acquisition of genetic novelty in genome evolution, we must also consider the impact of their own transcriptional activity. Results: We studied impact of TE transcriptional activity on gene using high-throughput RNA-Seq sequencing in Drosophila melanogaster. TEs, which turn out to be expressed in euchromatin as well as in heterochromatin, interact with genes at different levels. The observed transcription from TEs involve canonical or non-canonical transcription start sites (TSSs) distributed along their sequence. We also find evidences for potential bidirectional transcription from the TE promoter regions where the antisense transcript is co-opted by the host genome as TSSs of a gene. We found that active TEs seem to accumulate in the 5' upstream regions of the genes, and possibly provide an alternative transcript of the nearby gene. Indeed, predominantly, the TE transcript is collinear and overlapping the gene. Apart from the 5' upstream regions, we also found that most active TEs are transcribed on the gene transcript strand. Conversely, few transcripts from TE are anti sense with respect to the gene. This suggests that they have a disruptive action and are counter-selected. The only exceptions are for TEs located into introns, where they could provide another complex way of gene regulation, and in the 3' downstream region, where other mechanisms akin to siRNAs could take place. Finally, we noted several cases where the cryptic TSS is located on TE fragments corresponding to a low complexity sequence. Frequently these TE fragments appear to be over-represented when close to genes, suggesting a possible selected role. Conclusion: Altogether, these results suggest that active transposable elements influence host gene transcription. It is likely that some features of transposable elements have been exaptated in order to enrich the genes repertoire by opening routes to sub- or neo-functionalization. Examination of the transcription produced by transposable elements in D. melanogaster
Project description:The maintenance of genome integrity is an essential trait to the successful transmission of genetic information. In animal germ cells, piRNAs guide PIWI proteins to silence transposable elements (TEs) in order to maintain genome integrity. In insects, most of TE silencing in the germline is achieved by secondary piRNAs that are produced by a feed-forward loop (the ping-pong cycle), which requires the piRNA-directed cleavages of two types of RNAs: mRNAs of functional euchromatic TEs and heterochromatic transcripts that contain defective TE sequences. The first cleavage which initiates such amplification loop remains poorly understood. Taking advantage of the existence of strains that are devoid of functional copies of the LINE-like I-element, we report that in such Drosophila ovaries, the initiation of a ping-pong cycle is achieved only by secondary I-element piRNAs that are produced in the ovary and deposited in the embryonic germline. This unusual secondary piRNA biogenesis, detected in the absence of functional I-element copies, results from the processing of sense and antisense transcripts of several different defective I-elements. Once acquired, for instance after ancestor aging, this capacity to produce heterochromatic-only secondary piRNAs is partially transmitted through generations via maternal piRNAs. Furthermore, such piRNAs acting as ping-pong initiators in a chromatin-independent manner confer to the progeny a high capacity to repress the I-element mobility. Our study explains at the molecular level the basis for epigenetic memory of maternal immunity that protects females from hybrid dysgenesis caused by transposition of paternally inherited functional I-elements. Comparison of Drosophila small RNA populations in ovaries and/or eggs from 3-day-old or 25-day-old females.
Project description:Transposable elements (TEs), whose propagation can result in severe damage to the host genome, are silenced in the animal gonad by Piwi-interacting RNAs (piRNAs). piRNAs produced in the ovaries are deposited in the embryonic germline and initiate TE repression in the germline progeny. Whether the maternally transmitted piRNAs play a role in the silencing of somatic TEs is, however, unknown. Here we show that maternally transmitted piRNAs from the tirant retrotransposon in Drosophila are required for the somatic silencing of the TE and correlate with an increase in histone H3K9 trimethylation an active tirant copy. Comparison of tirant piRNAs in two Drosophila simulans natural populations.
Project description:Redox proteomics identifies additional putative targets of Sod1 redox regulation. The high abundance and broad cellular distribution of Sod1 suggests that it may function as a redox regulator of a broad variety of substrates in addition to GAPDH. To test this, we conducted a high-powered quantitative redox proteomics screen of WT and sod1Δ yeast to identify Sod1-dependent redox substrates. We used a combined SILAC-TMT approach, whereby sod1Δ-dependent changes in protein abundance are quantified through Stable Isotope Labeling with Amino acids in Cell culture (SILAC) and changes in reversible cysteine oxidation are quantified through cysteine-reactive Iodoacetyl Tandem Mass Tags (iodo-TMT) before and after reduction with DTT. Consequently, the study reveals a broad range of proteins that undergo significant changes in abundance, cysteine oxidation, or both. Focusing first on the effects of SOD1 deletion on proteome-wide protein abundance, we independently analyzed the SILAC MS data. A total of 4,409 proteins were confidently detected and quantified, and ~9% of these (373) exhibited significant differences in abundance between the two strains. Of these, 114 (30.6%) exhibit a significant decrease in protein abundance in sod1Δ cells compared to 259 (69.4%) proteins that exhibit a significant increase in abundance. Overall, we revealed a larger network of cysteine-containing proteins that are oxidized in a Sod1- dependent manner using mass spectrometry-based redox proteomics approaches.
Project description:The kidney contains distinct glomerular and tubulointerstitial compartments with diverse cell types and extracellular matrix components. Glomerular disease is characterized by excess matrix deposition and the loss of filtration barrier integrity. The role of immune cells in glomerular disease is crucial for dampening inflammation and maintaining homeostasis. Macrophages are innate immune cells influenced by their tissue microenvironment, but it is unclear how these cells contribute to distinct extracellular matrix compartments within the kidney. Bulk RNA-sequencing was used to determine the transcriptional landscapes of kidney macrophages.
Project description:Polycistronic mRNAs transcribed from operons are resolved via the trans-splicing of a spliced leader (SL) RNA. The SL is also frequently trans-spliced to monocistronic transcripts. Using a modified cap analysis of gene expression (CAGE) protocol we mapped sites of SL trans-splicing genome-wide in the marine chordate Oikopleura dioica and find evidence for proposed functions of SL-trans-splicing. A recent hypothesis postulates that operons facilitate recovery from growth arrested states in metazoans. We examined the expression dynamics of operons across the life-cycle of the animal and during growth arrest recovery. We show that operons do not facilitate recovery from growth arrest in O. dioica. We find that operons are enriched in the germline and that trans-spliced transcripts are predominantly maternal., Interestingly, there is a TOP-like motif in the SL sequence, and trans-splicing in TOP mRNAs, indicating that trans-spliced mRNAs are targets for nutrient-dependent translational control in O. dioica. Total RNA from a number of stages across development were pooled and used in a modified DeepCAGE protocol. A custom designed spliced-leader primer (using the SL exon) was used in the 2nd strand synthesis step.