Project description:The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSC), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was followed by a second substantial rewiring of transcriptional networks occurring in the trajectory to manifest leukaemia. We also find that both HSC and lineage-restricted granulocyte macrophage progenitors (GMP) acquired leukaemic stem cell (LSC) potential being capable of initiating and maintaining the disease. Finally, our data demonstrate that long-term expression of AE induces an indolent myeloproliferative (MPD)-like myeloid leukaemia phenotype with complete penetrance and that acute inactivation of AE function is a potential novel therapeutic option.
Project description:DNA methylation and histone H3 lysine 9 trimethylation (H3K9me3) play important roles in silencing of genes and retroelements. However, a comprehensive comparison of genes and repetitive elements repressed by these pathways has not been reported. Here we show that in mouse embryonic stem cells (mESCs), the genes up-regulated following deletion of the H3K9 methyltransferase Setdb1 are distinct from those de-repressed in mESC deficient in the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b, with the exception of a small number of primarily germline-specific genes. Numerous endogenous retroviruses (ERVs) lose H3K9me3 and are concomitantly de-repressed exclusively in SETDB1 knockout mESCs. Strikingly, ~15% of up-regulated genes are induced in association with de-repression of promoter proximal ERVs, half in the context of "chimaeric" transcripts that initiate within these retroelements and splice to genic exons. Thus, SETDB1 plays a previously unappreciated yet critical role in inhibiting aberrant gene transcription by suppressing the expression of proximal ERVs. NChIP-seq and mRNA-seq of WT, SETDB1 KO and DMNT1 TKO mESCs
Project description:HM1, HP1a-/-, and HP1b-/- ESC transcriptomes were generated to determine whether depletion of these HP1 proteins influences gene and/or retroelement expression mRNA profiles of HP1a and HP1b Knockouts and its corresponding wildtype
Project description:Purpose: The goal is to study the regulatory role of ferric uptake regulator (Fur) during anaerobic respiration in Shewanella piezotolerans WP3 mRNA profiles of WP3 wild type (WP3) and the fur mutant (Fur) were generated by deep sequencing using Illumina HiSeq 2000.
Project description:Abstract: Alternative splicing (AS) plays a major role in the generation of proteomic diversity and in gene regulation. However, the role of the basal splicing machinery in regulating AS remains poorly understood. Here we show that the core snRNP protein SmB/B’ self-regulates its expression by promoting the inclusion of a highly-conserved alternative exon in its own pre-mRNA that targets the spliced transcript for nonsense-mediated mRNA decay (NMD). Depletion of SmB/B’ in human cells results in reduced levels of snRNPs and in a striking reduction in the inclusion levels of hundreds of alternative exons, with comparatively few effects on constitutive exon splicing levels. The affected alternative exons are enriched in genes encoding RNA processing and other RNA binding factors, and a subset of these exons also regulate gene expression by activating NMD. Our results thus demonstrate a role for the core spliceosomal machinery in controlling an exon network that appears to modulate the levels of many RNA processing factors. HeLa cells were transfected with a control non-targeting siRNA pool (siNT), or with siRNA pools designed to knockdown SmB/B' or SRSF1 (also known as SF2/ASF/SFRS1). Sequence reads were aligned to exon-exon junction sequences in a database of EST/cDNA-mined cassette-type alternative splicing events. Processed data files (.bed and .txt) provided as supplementary files on the Series record. Processed data file build information: hg18.
Project description:Strand specific RNA sequencing of S. pombe revealed a highly structured programme of ncRNA expression at over 600 loci. Waves of antisense transcription accompanied sexual differentiation. A substantial proportion of ncRNA arose from mechanisms previously considered to be largely artefactual, including improper 3’ termination and bi-directional transcription. Constitutive induction of the entire spk1+, spo4+, dis1+ and spo6+ antisense transcripts from an integrated, ectopic, locus disrupted their respective meiotic functions. This ability of antisense transcripts to disrupt gene function when expressed in trans suggests that cis production at native loci during sexual differentiation may also control gene function. Consistently, insertion of a marker gene adjacent to the dis1+ antisense start site mimicked ectopic antisense expression in reducing the levels of this microtubule regulator and abolishing the microtubule-dependent “horsetail” stage of meiosis. Antisense production had no impact at any of these loci when the RNAi machinery was removed. Thus, far from being simply ‘genome chatter’, this extensive ncRNA landscape constitutes a fundamental component in the controls that drive the complex programme of sexual differentiation in S. pombe. Thorough interrogation of the Schizosaccharomyces pombe transcriptome during sexual differentiation using strand-specific total RNA sequencing (AB SOLiD 3.0 and 3.0+). A total of 19 samples were analysed by two separate machine runs (henceforth first and second runs, respectively). In the first machine run the following 5 samples were processed (on a single sequencing slide): Vegetative haploid (strain IH5974), pat1.114 diploid (IH2912) at vegetative growth (0) and pat1.114 diploid (IH2912) at 3, 5 and 10 hours following temperature shift from 25ºC to 32ºC to induce meiosis by Pat1 inactivation. In the second machine run the following 14 samples were processed (on two sequencing slides): Vegetative haploid (IH5974), pat1.114 diploid (IH2912) at vegetative growth (0) and pat1.114 diploid (IH2912) at 3, 5 and 10 hours following the temperature shift (a biological replicate of the first machine run). In addition, asynchronous IH3365 (wild type diploid) was also sequenced to enable a series of pair-wise haploid/diploid comparisons between itself, asynchronous haploid (IH5974) and pat1.114 diploid (IH2912) at vegetative growth. Finally, to find putative targets of the two bzip transcription factors atf21 and atf31, we sequenced RNA extracts from IH8832 (atf21.delta diploid) and IH8814 (atf31.delta diploid) before (0), and 3, 5, and 10 hours after the temperature shift, while the pat1.114 diploid (IH2912) at vegetative growth (0) and pat1.114 diploid (IH2912) at 3, 5 and 10 hours following the temperature shift were used as reference for this analysis.
Project description:The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA to make mature message. Schizosaccharomyces pombe Cwf10 (homolog Saccharomyces cerevisiae Snu114 and of Human U5-116K), an integral member of the U5 snRNP, is a GTPase that shares sequence homology with the eukaryotic translation elongation factor EF2. Cwf10 is required for pre-mRNA splicing; however, its mechanism(s) of action is still not understood. Cwf10/Snu114 family members contain a conserved N-terminal extension (NTE) that lacks homology with EF2 and has been predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing at all temperatures. Genetic interactions between cwf10-M-NM-^TNTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation. Characterization of Cwf10-NTE by various biophysical techniques shows the NTE contains both regions of structure and disorder in solution. The first twenty-three highly-conserved amino acids of the NTE are essential for its role in splicing, but are not sufficient to restore pre-mRNA splicing to wild-type levels in cwf10-M-bM-^HM-^FNTE cells. When the NTE is overexpressed in the cwf10-M-NM-^TNTE background, it can complement the truncated Cwf10 protein in trans, and it also immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of making specific contacts within the spliceosome that may facilitate Cwf10M-bM-^@M-^Ys overall role facilitating spliceosome rearrangements. Interrogation of the S. pombe transcriptome using poly-A enriched RNA sequencing (Illumina HiSeq 2500) in wild type and cwf10-M-NM-^TNTE cultures. A total of 4 samples were analysed: two biological repeats of wild-type strain and two biological repeats of cwf10-M-NM-^TNTE
Project description:Interleukin-15 (IL-15) and IL-2 possess distinct immunological functions despite both signaling through IL-2Rβ and the common cytokine receptor γ-chain, γc, We find that in the IL-15/IL-15Rα/IL-2Rβ/γc quaternary complex structure, IL-15 heterodimerizes IL-2Rβ and γc identically to the IL-2/IL-2Rα/IL-2Rβ/γc complex, despite differing receptor-binding chemistries. IL-15Rα dramatically increases the affinity of IL-15 for IL-2Rβ, and this allostery is required for IL-15 trans-signaling versus IL-2 cis-signaling. Consistent with the identical IL-2Rβ/γc dimer geometry, IL-2 and IL-15 exhibited similar signaling properties in lymphocytes, with any differences resulting from disparate receptor affinities. Thus, IL-15 and IL-2 induce similar signals, and the cytokine-specificity of IL-2Rα versus IL-15Rα determines cellular responsiveness. These results provide important new insights for specific development of IL-15- versus IL-2-based immunotherapeutics. RNA-Seq is conducted in mouse CD8+ T cells, not treated or treated with IL2 or IL15 for indicated concentrations (1nM or 500nM) and times (4hr or 24hr).
Project description:We present a comprehensive transcriptome of ciliate T. thermophila using the Illumina RNA-seq platform. The data was generated from the six mRNA samples of growth, starvation and conjugation of Tetrahymena. Despite an AT rich genome, there are about 124.6 million reads mapped to T. thermophila genome. Using these mapped reads, we have significantly improved the previous genome annotation and investigated the gene expression. Besides, our result also provided a comprehensive understanding of the alternative splicing in T. thermophila, and suggested the existence of the regulated unproductive splicing and translation (RUST) in the single-celled eukaryote. RNA-seq for six samples of Tetrahymena growth, starvation and conjugation.