Genetic fate mapping was preformed on aP2-Cre;tdTomato and aP2-Cre;tdTomato;SmoM2/+ animals and endothelial progenitor cells identified as the cell of origin of FN-RMS in aP2-Cre;SmoM2/+ animals
Hedgehog Pathway Drives Fusion-Negative Rhabdomyosarcoma Initiated From Non-myogenic Endothelial Progenitors.
Specimen part
View SamplesCdc34 is an essential E2 ubiquitin conjugating enzyme found in nearly all eukaryotes. It contains a highly conserved motif composed of S73/S97/12 amino acid insert near the active site cysteine. This motif is unique to Cdc34/Ubc7 type E2s while other E2s contain K/D/no insert at these positions. To better understand the function of this motif we mutated Cdc34 S73/S97/insert to be K/D/no insert and observed changes in transcript levels in mid-log phase yeast cells. ABSTRACT [Cdc34 is a ubiquitin conjugating enzyme necessary for the ubiquitylation of substrates by the SCF family of ubiquitin ligases. Previous work has shown that the Cdc34 protein is phosphorylated in vivo on serine residues. Cdc34 contains two serines within its catalytic domain, S73 and S97, that together with a 12 amino acid acidic loop, constitute a highly conserved motif (serine, serine, insert) among all members of the Cdc34 family of E2 enzymes. Using phosphospecific antibodies, we show that the essential serine S97 is indeed phosphorylated in vivo. Furthermore, this phosphorylation event is regulated by treatment with pheromone in yeast. Consistently, expression of a Cdc34 mutant lacking this motif (serine, serine, insert) leads to misregulation of the SCF substrates, Sic1, Far1, Cln1 and Cln2 and suppresses the cell cycle arrest brought about by an activated mating pathway. We further explored the function of this motif by microarray analysis and show that the transcripts of nearly the entire Sic1 cluster of co-transcribed genes is altered in a strain the expresses Cdc34 lacking this motif. Our data reveals that this highly conserved motif in Cdc34 and its phosphorylation are important for modulating SCF substrate abundance both transcriptionally and post-transcriptionally.]
New insight into the role of the Cdc34 ubiquitin-conjugating enzyme in cell cycle regulation via Ace2 and Sic1.
No sample metadata fields
View SamplesNine accessions of Arabidopsis were sampled before and after 14d of cold acclimation at 4°C. Transcript data were combined with metabolite data and related to quantitative measurement of plant freezing tolerance as determined by leaf electrolyte leakage assays.
Natural genetic variation of freezing tolerance in Arabidopsis.
Specimen part
View SamplesPurpose: We performed RNA-Immunoprecipitation in Tandem (RIPiT) experiments against human Staufen1 (Stau1) to identify its precise RNA binding sites in a transcriptome-wide manner. To monitor the consequences of Stau1 binding in terms of target mRNA levels and ribosome occupancy, we modified the levels of endogenous Stau1 in cells by siRNA or overexpression and performed RNA-sequencing and ribosome-footprinting experiments. Staufen1 (Stau1) is a double-stranded RNA (dsRNA) binding protein implicated in mRNA transport, regulation of translation, mRNA decay and stress granule homeostasis. Here we combined RNA-Immunoprecipitation in Tandem (RIPiT) with RNase footprinting, formaldehyde crosslinking, sonication-mediated RNA fragmentation and deep sequencing to map Staufen1 binding sites transcriptome-wide. We find that Stau1 binds complex secondary structures containing multiple short helices, many of which are formed by inverted Alu elements in annotated 3''UTRs or in "strongly distal" 3''UTRs extending far beyond the canonical polyadenylation signal. Stau1 also interacts with both actively translating ribosomes and with mRNA coding sequences (CDS) and 3''UTRs in proportion to their GC-content and internal secondary structure-forming propensity. On mRNAs with high CDS GC-content, higher Stau1 levels lead to greater ribosome densities, suggesting a general role for Stau1 in modulating the ability of ribosomes to elongate through secondary structures located in CDS regions. Overall design: We used HEK293 cells expressing near endogenous levels of wild-type Flag-Stau1 (65KDa isoform with an N-Terminal Flag tag). As a control we used a mutant version of Stau1 that is not functional for dsRNA binding. Formaldehyde crosslinking experiments and RNase footprinting experiments were done in two biological replicates. All RNASeq, Ribosome footprinting and PAS-Seq were done in two biological replicates.
Staufen1 senses overall transcript secondary structure to regulate translation.
No sample metadata fields
View SamplesThis experiment was set up in order to identify the (direct) transcriptional targets of the Ethylene Response Factor 115 (ERF115) transcription factor. Because ERF115 expression occurs in quiescent center (QC) cells and strong effects on the QC cells were observed in ERF115 overexpression plants, root tips were harvested for transcript profiling in order to focus on root meristem and QC specific transcriptional targets.
ERF115 controls root quiescent center cell division and stem cell replenishment.
Age, Specimen part
View SamplesAdipocytes arise from commitment and differentiation of adipose precursors in white adipose tissue (WAT). In studying adipogenesis, precursor markers, including Pref-1 and PDGFRa, are used to isolate precursors from stromal vascular fraction of WAT, but the relationship among the markers is not known. Here, we used Pref-1 promoter-rtTA system in mice for labeling Pref-1+ cells and for inducible inactivation of Pref-1 target, Sox9. We show requirement of Sox9 for maintenance of Pref-1+ proliferative, early precursors. Upon Sox9 inactivation, these Pref-1+ cells become PDGFRa+ cells that express early adipogenic markers. Thus, we show for the first time that Pref-1+ cells precede PDGFRa+ cells in the adipogenic pathway and that Sox9 inactivation is required for WAT growth and expansion. Furthermore, we show that, in maintaining early adipose precursors, Sox9 activates Meis1 which prevents adipogenic differentiation. Our study also demonstrates the Pref-1 promoter-rtTA system for inducible gene inactivation in early adipose precursor population. Overall design: RNA-Sequencing for differentially expressed genes (more than 2-fold) between GFP+ (Pref-1+) ingWAT SVF cells from floxed and Sox9 PreASKO mice (n=6 pooled).
Sox9-Meis1 Inactivation Is Required for Adipogenesis, Advancing Pref-1<sup>+</sup> to PDGFRα<sup>+</sup> Cells.
Sex, Age, Specimen part, Subject
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Sox9-Meis1 Inactivation Is Required for Adipogenesis, Advancing Pref-1<sup>+</sup> to PDGFRα<sup>+</sup> Cells.
Sex, Age, Specimen part, Cell line
View SamplesGene expression studies from hematopoietic stem cell (HSC) populations purified to variable degrees have defined a set of stemness genes. The present study describes the construction and comparative molecular analysis of l-phage cDNA libraries from highly purified primitive HSCs (PHSCs) which retained their long term repopulating activities (LTRAs), and from maturing HSCs (MHSCs) which were largely depleted of LTRAs. Library inserts were amplified and tagged by a T7 RNA polymerase promoter and used to generate biotinylated cRNA for Microarray hybridization. Microarray analysis of the libraries confirmed previous results but also revealed an unforseen preferential expression of translation and metabolism associated genes in the PHSCs. Therefore these data indicate that HSCs are quiescent only in regard of proliferative activities, but are in a state of readiness to provide the metabolic and translational activities required following induction of proliferation by factors which induce differentiation and exit from the HSC pool.
Gene expression profiles in murine hematopoietic stem cells revisited: analysis of cDNA libraries reveals high levels of translational and metabolic activities.
No sample metadata fields
View SamplesTranscriptome comparisons by RNAseq of genetically engineered mouse models of synovial sarcoma, expressing SS18-SSX1 or SS18-SSX2 and having homozygous conditional genetic silencing of Pten or wildtype Pten. Overall design: 6 primary tumor samples with wildtype Pten, 8 primary tumor and 5 metastatic site tumor samples with homozygous disruption of Pten
Epigenetic Changes at the <i>Birc5</i> Promoter Induced by YM155 in Synovial Sarcoma.
Subject
View SamplesThe first embryonic cell divisions rely on maternally stored mRNA and proteins. The zygotic genome is initially transcriptionally silenced and activated later in a process called zygotic genome activation (ZGA). ZGA in any species is still a poorly understood process; the timing of transcription onset is controversial and the identity of the first transcribed genes unclear. Zebrafish, Danio rerio, is a rapidly developing vertebrate model, which is accessible to experimentation and global studies before, during and after ZGA. Overall design: To accurately determine the onset of ZGA and to identify the first transcripts in zebrafish, we developed a metabolic labeling method, utilizing the ribonucleotide analog 4-thio-UTP, which allows efficient and specific affinity purification of newly transcribed RNA. Using deep sequencing, we characterized the onset of transcription in zebrafish embryos at 128-, 256-, and 512-cell stages. We identified 592 nuclear-encoded zygotically transcribed genes, comprising 670 transcript isoforms. Mitochondrial genomes were highly transcribed at all time points. Further, bioinformatic analysis revealed an enrichment of transcription factors and miRNAs among the newly transcribed genes, suggesting mechanistic roles for the early genes that are required to activate subsequent gene expression programs in development. Interestingly, analysis of gene-architecture revealed that zygotically transcribed genes are often intronless and short, reducing transcription and processing time of the transcript. The newly generated dataset enabled us to compare zygotically transcribed genes over a broad phylogenetic distance with fly and mouse early zygotic genes. This analysis revealed that short gene length is a common characteristic for early zygotically expressed genes. However, we detected a poor level of overlap for shared orthologs.
The earliest transcribed zygotic genes are short, newly evolved, and different across species.
No sample metadata fields
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