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SRP078437
Homo sapiens
2 Downloadable Samples
NextSeq 500
Description
The human genome contains approximately 27,700 CpG islands (CGIs). Most are associated with promoters and their DNA is nearly always unmethylated. By contrast, CGIs lying within the bodies of genes usually become methylated during differentiation and development. CGIs also normally become methylated at X-inactivated and imprinted genes and abnormally methylated in genome rearrangements and in malignancy. In such circumstances, methylation of CGIs is often associated with RNA transcripts reading through these elements but the relationship of this RNA to methylation of CGIs is not clear. Here we investigated a previously described form of a-thalassemia caused by a genome rearrangement leading to abnormal transcription and DNA methylation of the CGI at the promoter of the a-globin gene. We show that transcription per se is responsible for DNMT3B-mediated methylation of the globin CGI, and that this is a general mechanism responsible for methylation of most intragenic CpG islands. Overall design: CapSeq was performed on day 7 in vitro differentiated EBs containing the human gene sequence of RHBDF1 with (RHBDF1+P; chr16:47,861-63,210, hg18) or without (RHBDF1-P; chr16:47,911-60,819, hg18) its promoter in the a recombination mediated cassette exchange (RMCE) system established within the mouse a-globin locus (Lynch et al., 2012, DOI: 10.1038/emboj.2011.399 ) to map transcription initiation sites within the transgene. Please note that the Cap-seq methods captures the 5' end of any short RNA that was Capped, capturing both coding and non-coding RNA.
Publication Title
DNA methylation of intragenic CpG islands depends on their transcriptional activity during differentiation and disease.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Homo sapiens
- 113 Downloadable Samples
Affymetrix Human Genome U133A Array (hgu133a)
Description
In the present investigation, we have exploited the opportunity provided by neoadjuvant treatment of a group of postmenopausal women with large operable or locally advanced breast cancer (in which therapy is given with the primary tumour remaining within the breast) to take sequential biopsies of the same cancers before and after 10-14 days treatment with letrozole. RNA extracted from the biopsies has been subjected to Affymetrix microarray analysis and the data from paired biopsies interrogated to discover genes whose expression is most influenced by oestrogen deprivation.
Publication Title
Changes in breast cancer transcriptional profiles after treatment with the aromatase inhibitor, letrozole.
Sample Metadata Fields
No sample metadata fields
View Samples- Saccharomyces cerevisiae
- 12 Downloadable Samples
- Illumina HiSeq 2000
Description
Haploid budding yeast has two mating types, defined by the alleles of the MAT locus, MATa and MATa. Mating occurs when two haploid cells of opposite mating types signal to each other using reciprocal pheromones and receptors, polarize and grow towards each other, and eventually fuse to form a single diploid cell. The pheromones and receptors are necessary and sufficient to define a mating type, but other mating type-specific proteins make mating more efficient. We examined the role of these proteins by genetically engineering “transvestite” cells that swap the pheromone, pheromone receptor, and pheromone processing factors of one mating type for another. These cells can mate with each other, but their mating is inefficient. By characterizing their mating defects and examining their transcriptomes, we found Afb1 (a-factor barrier), a novel MATa-specific protein that interferes with a-factor, the pheromone secreted by MATa cells. We show that strong pheromone secretion is essential for efficient mating and that the weak mating of transvestites can be improved by boosting their pheromone production. Using synthetic biology, it is possible to characterize the factors that control efficiency in biological processes. In the case of budding yeast mating, selection for increased mating efficiency is likely to have continually boosted pheromone levels and the ability to discriminate between partners who make more (potentially fitter) and less (potentially less fit) pheromones. This sensitivity to which partner makes more pheromone comes at a cost: it means mating is not robust in situations where all potential partners make less pheromone. Overall design: 4 conditions were analysed, each with 3 biological replicates. The conditions were unstimulated MATa cells in YPD. Stimulated MATa cells in YPD+10nM a-factor. Unstimulated MATa cells in YPD. Stimulated MATa cells in YPD+10nM a-factor.
Publication Title
Genetically engineered transvestites reveal novel mating genes in budding yeast.
Sample Metadata Fields
Cell line, Subject
View Samples- Mus musculus
- 23 Downloadable Samples
Affymetrix HT MG-430 PM Array Plate (htmg430pm)
Description
Recent observations about how cells sense amino acids have argued for preeminent roles of mTOR and the stress kinase GCN2 in allowing cells to estimate their amino acid needs. Here we used models of programmed immune microenvironments where helper T cells have to sense how much amino acids are available to engage in antigen-fueled proliferation. Contrary to current models, T cells activate mTOR in the competency phase of the cell cycle regardless of amino acid amounts, GCN2 or surface TCR. Instead, we found T cells use an amino acid sensing system to target IL-2-induced STAT5 phosphorylation at the restriction point of cell cycle commitment. mTOR activity is subsequently reduced and specifically connected to SREBP activation. T cells can be pushed into cycle by increasing IL-2 even when no amino acids are available. Collectively, our studies reveal helper T cells use sequential and distinct pathways to measure local amino acid concentrations.
Publication Title
Proliferating Helper T Cells Require Rictor/mTORC2 Complex to Integrate Signals from Limiting Environmental Amino Acids.
Sample Metadata Fields
Specimen part, Treatment
View Samples- Mus musculus
- 12 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
The human and mouse aryl hydrocarbon receptor (hAHR and mAHRb) share limited (58%) transactivation domain sequence identity. Compared to the mAHRb allele, the hAHR displays 10-fold lower relative affinity for prototypical ligands such as 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). However, in previous studies we have demonstrated that the hAHR can display a higher relative ligand binding affinity than the mAHRb for specific AHR ligands such as indirubin. Each receptor has also been shown to differentially recruit LXXLL co-activator-motif proteins and to utilize different TAD subdomains in gene transactivation. Using hepatocytes isolated from C57BL6/J mice (Ahrb/b) and AHRTtr transgenic mice which express hAHR protein specifically in hepatocytes, we investigated whether the hAHR and mAHRb differentially regulate genes. Microarray and quantitative-PCR analysis of Ahrb/b and AHRTtr primary-mouse hepatocytes treated with 10 nM TCDD revealed that a number of established AHR target genes such as Cyp1a1 and Cyp1b1 are significantly induced by both receptors. Remarkably, of the 1752 genes induced by mAHRb and 1186 genes induced by hAHR, only 265 genes (<10%) were significantly activated by both receptors in response to TCDD. Conversely of the 1100 and 779 genes significantly repressed in mAHRb and hAHR hepatocytes respectively, only 462 (<25%) genes were significantly repressed by both receptors in response to TCDD treatment. Genes identified as differentially expressed are known to be involved in a number of biological pathways, including cell proliferation and inflammatory response which suggests that compared to the mAHRb, the hAHR may play contrasting roles in TCDD-induced toxicity and endogenous AHR-mediated gene regulation.
Publication Title
Differential gene regulation by the human and mouse aryl hydrocarbon receptor.
Sample Metadata Fields
Specimen part
View Samples- Macaca mulatta
- 10 Downloadable Samples
- Affymetrix Human Genome U133A Array (hgu133a)
Description
Nuclei of the mammalian thalamus are aggregations of neurons with unique architectures and input-output connections, yet the molecular determinants of their organizational specificity remain unknown. By comparing expression profiles of thalamus and cerebral cortex in adult rhesus monkeys we identified transcripts that are unique to dorsal thalamus or to individual nuclei within it. Real-time quantitative polymerase chain reaction and in situ hybridization analyses confirmed the findings. Expression profiling of individual nuclei microdissected from the dorsal thalamus revealed additional subsets of nucleus-specific genes. Functional annotation using Gene Ontology (GO) vocabulary and Ingenuity Pathway analysis revealed over-representation of GO categories related to development, morphogenesis, cell-cell interactions, and extracellular matrix within the thalamus- and nucleus-specific genes-many involved in the Wnt signaling pathway. Examples included the transcription factor TCF7L2, localized exclusively to excitatory neurons, a calmodulin-binding protein PCP4, the bone extracellular matrix molecules SPP1 and SPARC, and other genes involved in axon outgrowth and cell matrix interactions. Other nucleus-specific genes such as CBLN1 are involved in synaptogenesis. The genes identified likely underlie nuclear specification, cell phenotype and connectivity during development and their maintenance in the adult thalamus.
Publication Title
Nucleus- and cell-specific gene expression in monkey thalamus.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 6 Downloadable Samples
- Illumina HiSeq 2500
Description
Bone adaptation to mechanical loading is regulated via signal transduction by mechano-sensing osteocytes. Mineral-embedded osteocytes experience strain-induced interstitial fluid flow and fluid shear stress, and broad shifts in gene expression are key components in the signaling pathways that regulate bone turnover. RNA sequencing analysis, or RNA-Seq, enables more complete characterization of mechano-sensitive transcriptome regulation than previously possible. We hypothesized that RNA-Seq of osteocytic MLO-Y4 cells reveals both expected and novel gene transcript regulation in cells previously fluid flowed and analyzed using gene microarrays (Govey et al., J Biomech, 2014). MLO-Y4 cells were flowed for 2 h with 1 Pa oscillating fluid shear stress and post-incubated 2 h. RNA-Seq of original samples detected 58 fluid flow-regulated gene transcripts (p-corrected<0.05) versus 65 transcripts detected by microarray. However, RNA-Seq demonstrated greater dynamic range, with all 58 transcripts >1.5 fold-change whereas 10 of 65 met this cut-off by microarray. Analyses were complimentary in patterns of regulation, though only 6 transcripts were significant in both analyses: Cxcl5, Cxcl1, Zc3h12a, Ereg, Slc2a1, and Egln1. As part of a broad inflammatory response inferred by gene ontology analyses, we again observed greatest up-regulation of inflammatory C-X-C motif chemokines, and newly implicated HIF-1? and AMPK signaling pathways. Importantly, we detected both expected mechano-sensitive transcripts (e.g. Nos2, Ptgs2, Ccl7) and transcripts not previously identified as mechano-sensitive, e.g. Ccl2. We found RNA-Seq advantageous over microarrays because of its ability to analyze unbiased estimation of gene expression, informing our understanding of osteocyte signaling. Overall design: Osteocyte-like MLO-Y4 cells were subjected to 2 hours of 10 dyn/cm^2 oscillating fluid flow in parallel-plate fluid flow chambers and harvested for analysis after an additional 2 hours post-flow incubation in fresh medium. Parallel control samples from sham treated cells were also collected. Triplicate samples of both flow and non-flow control conditions were collected to analyze flow vs. non-flow gene transcript regulation.
Publication Title
Functional and structural characterization of osteocytic MLO-Y4 cell proteins encoded by genes differentially expressed in response to mechanical signals in vitro.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 6 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Although gain of chromosome-5p is one of the most frequent DNA copy number imbalances in cervical squamous cell carcinoma (SCC), the genes that drive its selection remain poorly understood. In a previous cross-sectional clinical study we showed that the microRNA processor Drosha (located on chromosome-5p) demonstrates frequent copy-number gain and over-expression in cervical SCC, associated with altered microRNA profiles. Here, we have conducted gene depletion/over-expression experiments to demonstrate the functional significance of up-regulated Drosha in cervical SCC cells. Drosha depletion by RNA-interference (RNAi) produced significant, specific reductions in cell motility/invasiveness in vitro, with a silent RNAi-resistant Drosha mutation providing phenotype rescue. Unsupervised hierarchical clustering following global profiling of 319 microRNAs in eighteen cervical SCC cell line specimens generated two groups according to Drosha expression levels. Altering Drosha levels in individual SCC lines changed the group into which the cells clustered, with gene depletion effects being rescued by the RNAi-resistant mutation. Forty-five microRNAs showed significant differential expression between the groups, including four of fourteen that were differentially-expressed in association with Drosha levels in clinical samples. miR-31 up-regulation in Drosha over-expressing samples/cell lines was the highest-ranked change (by adjusted p-value) in both analyses, an observation validated by Northern blotting. These functional data support the role of Drosha as an oncogene in cervical SCC, by affecting expression of cancer-associated microRNAs that have the potential to regulate numerous protein-coding genes.
Publication Title
Functional evidence that Drosha overexpression in cervical squamous cell carcinoma affects cell phenotype and microRNA profiles.
Sample Metadata Fields
Sex, Cell line
View Samples- Homo sapiens
- 6 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Comparison of mRNA expression profiles in W12 Series 1 cervical ectokeratinocytes at passage number 22 versus 19 (during which time the cells gain an invasive phenotype)
Publication Title
Functional evidence that Drosha overexpression in cervical squamous cell carcinoma affects cell phenotype and microRNA profiles.
Sample Metadata Fields
Sex, Cell line
View Samples- Homo sapiens
- 11 Downloadable Samples
- Affymetrix Human Genome U133A 2.0 Array (hgu133a2)
Description
Stable knockdown of NET1, a RhoGEF, was achieved in AGS Gastric Cancer cells. This gene is known to be overexpressed in the disease.
Publication Title
A functional and transcriptomic analysis of NET1 bioactivity in gastric cancer.
Sample Metadata Fields
Cell line
View Samples