Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide-encoding genes in vertebrates, providing an entry point to define their function in vivo. Overall design: Ribosome profiling experiments at five timepoints across zebrafish development in WT embryos
Upstream ORFs are prevalent translational repressors in vertebrates.
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To explore putative connections between genetic methionine restriction and the retrograde response, we asked whether the altered transcriptional program of methionine-restricted cells required RTG3 (which is indispensible for retrograde signaling in yeast).
Methionine restriction activates the retrograde response and confers both stress tolerance and lifespan extension to yeast, mouse and human cells.
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The human Werner and Bloom syndromes (WS and BS) are caused by deficiencies in the WRN and BLM RecQ helicases, respectively. WRN, BLM and their S. cerevisiae homologue Sgs1, are particularly active in vitro in unwinding G-quadruplex DNA (G4-DNA), a family of non-canonical nucleic acid structures formed by certain G-rich sequences. Recently, mRNA levels from loci containing potential G-quadruplex-forming sequences (PQS) were found to be preferentially altered in sgs1 mutants, suggesting that G4-DNA targeting by Sgs1 directly affects gene expression. Here, we extend these findings to human cells. Using microarrays to measure mRNAs obtained from human fibroblasts deficient for various RecQ family helicases, we observe significant associations between loci that are upregulated in WS or BS cells and loci that have PQS. No such PQS associations were observed for control expression datasets, however. Furthermore, upregulated genes in WS and BS showed no or dramatically reduced associations with sequences similar to PQS but that have considerably reduced potential to form intramolecular G4-DNA. These findings indicate that, like Sgs1, WRN and BLM can regulate transcription globally by targeting G4-DNA.
Altered gene expression in the Werner and Bloom syndromes is associated with sequences having G-quadruplex forming potential.
Sex, Age, RaceView Samples
In Xenopus laevis, a number of studies identified vegetal factors that specify the germ line, endoderm and dorsal axis, but there are few studies demonstrating roles for animal-enriched maternal mRNAs.
Novel animal pole-enriched maternal mRNAs are preferentially expressed in neural ectoderm.
Specimen partView Samples
Transcriptome analysis was conducted on vorinostat resistant HCT116 cells (HCT116-VR) upon knockdown of potential vorinostat resistance candidate genes in the presence and absence of vorinostat. Potential vorinostat resistance candidate genes chosen for this study were GLI1 and PSMD13, which were identified through a genome-wide synthetic lethal RNA interference screen. To understand the transcriptional events underpinning the effect of GLI1 and PSMD13 knockdown (sensitisation to vorinostat-induced apoptosis), cells were first subjected to gene knockdown, then to treatment with vorinsotat or the solvent control. Two timepoints for drug treatment were assessed: a timepoint before induction of apoptosis (4hrs for siGLI1 and 8hrs for siPSMD13) and a timepoint when apoptosis could be detected (8hrs for siGLI1 and 12hrs for siPSMD13). Overall design: There are 42 samples in total, from triplicate independent biological experiments of 14 samples each.
A genome scale RNAi screen identifies GLI1 as a novel gene regulating vorinostat sensitivity.
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BMP4 is down-regulated in metastatic human and murine mammary tumours. Here we determined the effect of ectopic mouse Bmp4 re-expression on global gene expression patterns in orthotopic primary mammary tumours in syngeneic Balb/c mice.
BMP4 inhibits breast cancer metastasis by blocking myeloid-derived suppressor cell activity.
Sex, Specimen partView Samples
The identification of recurrent somatic mutations in genes encoding epigenetic enzymes, coupled with biochemical studies demonstrating aberrant recruitment of epigenetic enzymes such as histone deacetylases (HDACs) and histone methyltransferases (HMTs) to promoter regions through association with oncogenic fusion proteins such as PML-RAR and AML1-ETO has provided a strong rationale for the development compounds that target the epigenome for the treatment of cancer. HDAC inhibitors (HDACi) are potent inducers of tumor cell apoptosis but it remains unclear why tumor cells are selectively sensitive to HDACi-induced cell death.
HDAC inhibitors induce tumor-cell-selective pro-apoptotic transcriptional responses.
Specimen part, TimeView Samples
Anti-retroviral therapy (ART) has transformed human immunodeficiency virus (HIV) infection from a fatal illness to a chronic condition by controlling viral replication and restoring immune function. However, chronic T-cell activation can be observed in 20-35% of individuals on ART, resulting in an immune reconstitution inflammatory syndrome (IRIS) [1-3]. IRIS involving the CNS can result in permanent disability and death . Tat is a viral protein produced in HIV-infected cells and released into the extracellular space . We show that the secreted-Tat protein activated uninfected T-cells in an antigen-independent manner without inducing proliferation. Notably, Tat induced the secretion of IL-17 from T-cells and increased the percentage of T-cells with a Th17 phenotype. T-cell activation was independent of the T-cell receptor but dependent on endocytosis of Tat and activation of vascular endothelial growth factor receptor 2 (VEGFR2). Tat induced global changes in histone acetylation and increased HIV infection in non-replicating T-cells. Furthermore, in an individual with CNS IRIS, Tat expressing infiltrates and secretion of IL-17 was detected in the absence of viral replication in the brain. Thus Tat can induce T-cell activation in a paracrine and autocrine manner resulting in propagation of inflammation and increased virulence.
Induction of IL-17 and nonclassical T-cell activation by HIV-Tat protein.
Specimen part, Treatment, TimeView Samples
Purpose: Breeding for gibberella ear rot resistance have been challenging due to the high complexity of the trait. The current study attempts to characterize defence responses to Fusarium graminearum infection in two maize inbred lines with different levels of resistance to the pathogen. Methods: RNA was extracted from developing kernels of two inbred lines, which had been either fungal (F. graminearum DAOM180378) or mock inoculated 11 days post sibcrossing, using a guanidine isothiocyanate method and ultra-centrifugation with cesium chloride. Isolated RNA was used to quantify whole genome gene expression using RNA-seq (Illumina TruSeq RNA library prep kit v2, Illumina HiSeq 2000). Paired end reads generated from RNA-seq were trimmed of adaptors and low quality reads, aligned with the B73 reference genome sequence version 2, expression levels (TPM) were computed and differential gene expression analysis were performed using CLC Genomics Workbench version 9. Results: Gene transcripts responding to fungal infection were captured by comparing gene expression levels in mock and fungal inoculated maize ears and gene ontology terms associated with significantly up-regulated gene transcripts were determined for each inbred. More genes were up regulated in the susceptible inbred relative to the resistant inbred, many of which are associated with oxidation-reduction processes potentially causing earlier programmed cell death in the susceptible inbred. Conclusions: This information helped to identify gene transcripts that were relevant in defense responses with potential applicability in routine breeding efforts and to propose an effective GER resistance mechanism. Overall design: The experiment consisted of 16 RNA samples from two inbreds (B73 and CO441) tested over two years (2004 and 2006), two treatments (mock and fungal) and two sampling times (1 and 2 days after inoculation).
Transcriptome profiling of two maize inbreds with distinct responses to Gibberella ear rot disease to identify candidate resistance genes.
Specimen part, Treatment, SubjectView Samples
Glioblastoma HSR-GBM1 cells have low mitochondrial DNA copy numbers which is associated with the abnormal DNA methylation patterns. By inducing DNA demthylation using 5azacytidine and vitamin C, HSR-GBM1 cells modulate their mitochondrial copy number and capability of differentiation. Overall design: HSR-GBM1 cell line with three conditions: untreated control group, treated with 5 azacitidine and treated with vitamin C. 3 biological replicates of each condition.
Global DNA methylation synergistically regulates the nuclear and mitochondrial genomes in glioblastoma cells.
Specimen part, Cell line, SubjectView Samples