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SRP069174
Mus musculus
12 Downloadable Samples
Illumina HiSeq 2000
Description
Inactivating mutations in the zinc finger gene PHF6 are seen in approximately 40% of adult T-cell acute lymphoblastic leukemias (T-ALLs) and 3% of adult acute myeloid leukemias (AMLs). The absence of PHF6 mutations in B-cell lineage malignancies has led to the hypothesis that PHF6 may act as a lineage-specific tumor suppressor gene. Here, we demonstrate that PHF6 plays a critical role in regulating B-cell identity in the context of B-cell precursor acute lymphoblastic leukemia (preB-ALL). Transplantation of Phf6 knockout preB-ALL cells (hereafter referred to as Phf6KO cells) into immunocompetent syngeneic recipients resulted in the development of a fully penetrant lymphoma-like disease. Strikingly, the resulting lymphomas showed robust up-regulation of the canonical T-cell marker CD4, suggesting that Phf6KO cells adopt a T-cell program in the context of leukemogenesis. RNA sequencing analysis revealed numerous differentially expressed (DE) genes in Phf6WT and Phf6KO cells, including a significant down-regulation of genes and gene sets involved in pathways important for B-cell development. Chromatin immunoprecipitation followed by high-throughput sequencing analysis revealed that PHF6 co-localizes with H3K27ac signals close to the transcription start sites (TSSs) and enhancer regions of a significant proportion of DE genes. Notably, regions flanking the TSS of DE genes showed significant enrichment for binding sites of several well-described master regulators of B-cell development, including PU.1, EGR-1, EBF-1, NF-kB, TCF3 and TCF12. We found that PHF6 and TCF12 physically interact in preB-ALL cells, suggesting that these factors act synergistically in the establishment and maintenance of B-cell identity. In addition, we found that a human PHF6 mutant T-ALL cell line has an incompletely rearranged IGH locus, strongly suggesting that T-ALL can have a B-cell origin. These findings reveal an essential role for PHF6 in the establishment and maintenance of B-cell identity in preB-ALL by directly activating genes that are crucial for B-cell lineage commitment and maintenance. Collectively, these results indicate that loss of function of PHF6 in preB-ALL leads to an unstable cellular state in which cells acquire alternate developmental programs (such as the T-lineage program) to survive, potentially explaining the apparent absence of PHF6 mutations in human B cell-lineage malignancies. Overall design: Gene expression profiles by RNA-Seq of 3 Phf6 wild-type preB-ALL cells, 3 shPhf6 preB-ALL cells, 6 Phf6 knockout (2 different sgRNAs) preB-ALL cells
Publication Title
PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Caenorhabditis elegans
- 30 Downloadable Samples
- Illumina HiSeq 2000
Description
HIV-1 nucleoside reverse transcriptase inhibitor (NRTI) use is associated with severe adverse events. However, the exact mechanisms behind their toxicity has not been fully understood. Mitochondrial dysfunction after chronic exposure to NRTIs has predominantly been assigned to mitochondrial polymerase-? inhibition by NRTIs. However, an increasing amount of data suggests that this is not the sole mechanism. Many NRTI induced adverse events have been linked to the incurrence of oxidative stress, although the causality of events leading to reactive oxygen species (ROS) production and their role in toxicity is unclear. In this study we show that short-term effects of these drugs, which are rarely discussed in the literature, include direct inhibition of the mitochondrial respiratory chain (MRC), decreased ATP levels and increased ROS production. Collectively these events affect fitness and longevity of C. elegans through mitohormetic signalling events. Furthermore, we demonstrate that these effects can be normalized by addition of the anti-oxidant N-acetylcysteine (NAC), which suggests that ROS likely influence the onset and severity of adverse events upon drug exposure. Overall design: RNA-seq on Caenorhabditis elegans exposed to DMSO, 3''-azido-3''-deoxythymidine (zidovudine or AZT), 2'',3''-didehydro-2'',3''-deoxythymidine (stavudine or d4T), 3''-deoxy-3''-fluorothymidine (alovudine or FLT) or untreated control after 24 or 72 hours of exposure.
Publication Title
Beyond the polymerase-γ theory: Production of ROS as a mode of NRTI-induced mitochondrial toxicity.
Sample Metadata Fields
Specimen part, Subject
View Samples- Mus musculus
- 17 Downloadable Samples
Affymetrix Mouse Gene 1.0 ST Array (mogene10st)
Description
These studies utilized two TCR transgenic mouse lines, LLO118 and LLO56, that our laboratory has developed and characterized, which recognize the same Listeria monocytogenes LLO/IO-Ab epitope with equal affinities. When 104 naive CD4+ LLO T cells are transferred into a B6 mouse and one day later infected with wild type Listeria monocytogenes, the LLO118 T cells have a more robust primary expansion than LLO56. In contrast, after a secondary challenge, LLO56 T cells have a much greater expansion than LLO118 T cells. One striking phenotypic difference between the LLO118 and LLO56 T cells lies in their CD5 expression. CD5 expression has been shown to correlate directly with TCR affinity for self-pMHC and tonic signaling. LLO56 cells have a higher basal phosphorylation of the TCR chain, and they have significantly increased expression of Nur77 mRNA. These transcriptional profiling experiments examined if there were transcriptional differences between LLO118 and LLO56 T cells, either naive or after D7 of infection, that would account for their disparate in vivo behaviors.
Publication Title
Tonic TCR Signaling Inversely Regulates the Basal Metabolism of CD4<sup>+</sup> T Cells.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 30 Downloadable Samples
- Affymetrix Human Exon 1.0 ST Array [transcript (gene) version (huex10st)
Description
Multiple myeloma (MM), an incurable plasma cell malignancy, requires localisation within the bone marrow in order to survive and proliferate. Interactions between the malignant plasma cell and bone marrow mesenchymal stem cell (BMMSC) are thought to be a critical determinant of this requirement, and include both physical and chemical components. There is increasing evidence that the phenotype of the BMMSC is stably altered in patients with MM. More recently, it has been suggested that this phenotypic transformation is also observed in patients with the benign condition known as monoclonal gammopathy of undetermined significance (MGUS), which almost always precedes MM. In this study, we describe a mechanism by which the peptidyl arginine deiminase 2 (PADI2) enzyme plays an key role in the control of malignant plasma cell phenotype by BMMSCs. PADI enzymes deiminate (citrullinate) peptidyl arginine residues, changing the function or interactions made by the target protein. We identified PADI2 as one of the most highly upregulated transcripts in BMMSCs from both MGUS and MM patients, and that through citrullination of arginine residue 26 of histone H3, it induces the upregulation of interleukin-6 (IL-6) expression. This directly leads to the acquisition of resistance to the chemotherapeutic agent, bortezomib, by malignant plasma cells. We therefore describe a novel mechanism by which BMMSC dysfunction in patients with MGUS and MM directly leads to pro-malignancy signalling through the citrullination of histone H3R26.
Publication Title
Citrullination of histone H3 drives IL-6 production by bone marrow mesenchymal stem cells in MGUS and multiple myeloma.
Sample Metadata Fields
Sex, Age, Specimen part, Disease, Disease stage
View Samples- Mus musculus
- 6 Downloadable Samples
- Affymetrix Mouse Gene 1.0 ST Array (mogene10st)
Description
Neurospheres generated in vitro were treated with non-epinephrine or potassium chloride. Gene expression analysis was then carried out to identify genes that are up or down regulated due to chemical treatement.
Publication Title
A comparative study of techniques for differential expression analysis on RNA-Seq data.
Sample Metadata Fields
Sex, Specimen part
View Samples- Mus musculus
- 29 Downloadable Samples
- Affymetrix Mouse Genome 430A 2.0 Array (mouse430a2)
Description
Activation of the MLL-ENL-ERtm oncogene initiates aberrant proliferation of myeloid progenitors. Here, we show induction of a fail-safe mechanism mediated by the DNA damage response (DDR) machinery that results in activation of the ATR/ATM-Chk1/Chk2-p53/p21 checkpoint and cellular senescence at early stages of cellular transformation caused by a regulatable MLL-ENL-ERtm in mice. Furthermore, we identified the transcription program underlying this intrinsic anti-cancer barrier, and DDR-induced inflammatory regulators that fine-tune the signaling towards senescence, thereby modulating the fate of MLL-ENL-immortalized cells in a tissue-environment-dependent manner. Our results indicate that DDR is a rate-limiting event for acquisition of stem cell-like properties in MLL-ENL-ERtm-mediated transformation, as experimental inhibition of the barrier accelerated the transition to immature cell states and acute leukemia development.
Publication Title
DNA damage response and inflammatory signaling limit the MLL-ENL-induced leukemogenesis in vivo.
Sample Metadata Fields
Specimen part, Disease stage
View Samples- Homo sapiens
- 14 Downloadable Samples
- Affymetrix Clariom S Human array (clariomshuman)
Description
Metastasis of human tumours to LNs is a universally negative prognostic factor. LN stromal cells (SCs) play a crucial role in enabling T cell responses, and since tumour metastases modulate their structure and function, this interaction may suppress immune responses to tumour antigens. However the SC subpopulations that respond to infiltration of malignant cells into human LNs have not been defined. Using microarray, we sought to assess gene expression profiles of two distinct SC subpopulations isolated from melanoma-infiltrated LNs.
Publication Title
Distinctive Subpopulations of Stromal Cells Are Present in Human Lymph Nodes Infiltrated with Melanoma.
Sample Metadata Fields
Specimen part
View Samples- Homo sapiens
- 8 Downloadable Samples
- Illumina HiSeq 2500
Description
Understanding the physiological relevance of structures in mammalian mRNAs remains elusive, especially considering the global unfolding of mRNA structures in eukaryotic organisms recently examined, as well as the decade-long observation that mRNAs generally seem no more likely than random sequences to be stably folded. Here we show that RNA secondary structures, mostly weak and close-to-random, facilitate the 3'-end processing of thousands of human mRNAs by juxtaposing poly(A) signals (PASs) and cleavage sites that are otherwise too far apart. Folding of these 3'-end structures also enhances mRNA stability. Global structure probing shows that 3'-end regions are indeed folded in cells despite substantial unfolding of PAS-upstream regions. Analyses of thousands of ectopically expressed variants prove that folding both enhances processing and increases stability. Mutagenesis of a genomic locus further implicates structure-controlled processing in regulating neighboring gene expression. These results reveal widespread roles for RNA structure in mammalian mRNA biogenesis and metabolism. Overall design: This series includes 8 samples designed to measure the efficiency of 3'' end processing from a reporter library expressed in HEK293T cells and HeLa cells, in steady state or in nascent RNAs (by 4sU labeling and capture).
Publication Title
Widespread Influence of 3'-End Structures on Mammalian mRNA Processing and Stability.
Sample Metadata Fields
Cell line, Subject
View Samples- Caenorhabditis elegans
- 2 Downloadable Samples
- Illumina Genome Analyzer
Description
Small endogenous C. elegans RNAs from L4 and young adult worms were prepared for sequencing using a protocol derived from Batista et al., (2008) and Lau et al. (2001). The small-RNA libraries were constructed using a method that does not require a 5' monophosphate (called 5' monophosphate-independent method, Ambros et al., 2003) to profile secondary siRNAs that have 5' triphosphorylated G. All preprocessed small-RNA reads were mapped to genome (ce6), allowing no mismatches. After excluding miRNAs, 21U RNAs, rRNAs, and other structural ncRNAs, the remaining reads were classified as 22G RNAs, 26G RNAs, and other siRNAs, based on their length and 5' terminal nucleotide. Overall design: Small-RNA libraries were sequenced in L4 and young adult stages in C.elegans.
Publication Title
Long noncoding RNAs in C. elegans.
Sample Metadata Fields
Cell line, Subject
View Samples- Homo sapiens
- 9 Downloadable Samples
- Illumina HiSeq 4000
Description
The development of CRISPR-Cas systems for targeting DNA and RNA in diverse organisms has transformed biotechnology and biological research. Moreover, the CRISPR revolution has highlighted bacterial adaptive immune systems as a rich and largely unexplored frontier for discovery of new genome engineering technologies. In particular, the class 2 CRISPR-Cas systems, which use single RNA-guided DNA-targeting nucleases such as Cas9, have been widely applied for targeting DNA sequences in eukaryotic genomes. Here, we report DNA-targeting and transcriptional control with class I CRISPR-Cas systems. Specifically, we repurpose the effector complex from type I variants of class 1 CRISPR-Cas systems, the most prevalent CRISPR loci in nature, that target DNA via a multi-component RNA-guided complex termed Cascade. We validate Cascade expression, complex formation, and nuclear localization in human cells and demonstrate programmable CRISPR RNA (crRNA)-mediated targeting of specific loci in the human genome. By tethering transactivation domains to Cascade, we modulate the expression of targeted chromosomal genes in both human cells and plants. This study expands the toolbox for engineering eukaryotic genomes and establishes Cascade as a novel CRISPR-based technology for targeted eukaryotic gene regulation. Overall design: Examination of transcriptome-wide changes in gene expression with Cascade-mediated activation of endogenous genes.
Publication Title
Targeted transcriptional modulation with type I CRISPR-Cas systems in human cells.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples