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:We made insulation (STITCH+30kb) and deletion (del(30-440)) alleles of the MYC enhancer in human iPS cells. We employed RNA-seq to see how the insulation and deletion affects the transcriptome of the cells. We prepared libraries from three replicates for each configuration.
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:The induction of genes in response to exposure of T. reesei to wheat straw was explored using genome-wide RNA-seq and compared to published RNA-seq data and model of how A. niger senses and responds to the lignocellulose. After 24 h of exposure to straw, transcript levels of known and predicted lignocellulose-degrading enzymes increased to around 8% of total cellular mRNA in T. reesei, which was much less when compared to A. niger. The bulk of enzymes used to deconstruct wheat straw is similar in both fungi. Other, non-plant cell wall-degrading enzymes which may aid in lignocellulose degradation were also uncovered in T. reesei and similar to those described in A. niger. Antisense transcripts were also shown to be present in T. reesei and their expession can be regulated by the respective growth condition. Triplicate samples of T. reesei cultivated in each of the three following conditions were taken: 1) After 48 h growth in glucose-based minimal media; 2) After transfer of mycelia from glucose-based media into media containing wheat straw as a sole carbon source and 3) 5 h after addition of glucose to straw cultures.
Project description:A molecular and bioinformatic pipeline permitting comprehensive analysis and quantification of myocardial miRNA and mRNA expression with next-generation sequencing was developed and the impact of enhanced PI3Kalpha signaling on the myocardial transcriptome signature of pressure overload-induced pathological hypertrophy was explored. miRNA and mRNA-Seq were carried out in four groups of mouse LV samples: WT sham, WT+TAC, caPI3Kalpha sham, caPI3Kalpha+TAC
Project description:Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In Study One, 270 predominantly Angus heifers were classified based on fertility using four rounds of serial embryo transfer (ET) to select animals with intrinsic differences in pregnancy loss. In each round, a single in vitro-produced high-quality embryo was transferred into heifers on day 7 post-estrus and pregnancy was determined on days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In Study Two, fertility-classified heifers were resynchronized and bred with semen from a single high fertility bull. Blood samples were collected every other day from days 0 to 36 post-mating. Pregnancy rate was determined on day 28 by ultrasound and tended to be higher in HF (70.4%) and SF (46.7%) than IF (0%) heifers. Progesterone concentrations in serum during the first 20 days post-estrus were not different in non-pregnant heifers and also not different in pregnant heifers among fertility groups. In Study Three, a single in vivo-produced embryo was transferred into fertility-classified heifers on day 7 post-estrus. The uteri were flushed on day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the day 14 endometrial biopsies of pregnant HF, SF and IF heifers (n=5 per group) and analyzed by edgeR robust analysis. There were 26 differentially expressed genes (DEG) in the HF compared to SF endometrium, 12 DEG for SF compared to IF endometrium, and 3 DEG between the HF and IF endometrium. Many of the DEG encoded proteins involved in immune responses and are expressed in B cells. Results indicate that pre-implantation conceptus survival and growth to day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between days 14 and 28 when pregnancy recognition signaling and conceptus implantation must occur for the establishment of pregnancy. Endometrial biopsies were subjected to RNA sequencing from high fertile (HF; n=5), subfertile (SF; n=5) and infertile (IF; n=5) classified heifers on day 14 of pregnancy.
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: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:The transcriptomic profiling of psoriasis has led to an increased understanding of disease pathogenesis. Although microarray technologies have been instrumental in this regard, it is clear that these tools detect an incomplete set of DEGs. RNA-seq can be used to supplement these prior technologies. Here, the use of RNAseq methods substantially increased the number of psoriasis-related DEGs. Furthermore, DEGs that were uniquely identified by RNA-seq, but not in other published microarray studies, further supported the role of IL-17 and tumor necrosis factor-a synergy in psoriasis. Examination of one of these factors at the protein level confirmed that RNA-seq is a powerful tool that can be used to identify molecular factors present in psoriasis lesions, and may be useful in the identification of therapeutic targets that to our knowledge have not been reported previously. Further studies are in progress to determine the biological significance of DEGs uniquely discovered by RNA-seq. To define the transcriptomic profile of psoriatic skin, three pairs of lesional and nonlesional skin biopsy specimens were taken from patients with untreated moderate-to-severe plaque psoriasis.