In this experiment, total RNA was extracted from asynchronous population of L1210 cells and hybridized to Affymetrix 430A 2.0 arrays in order to obtain an expression profile of these cells. We have previously mapped the replication timing of the entire mouse genome in this cell line, using mouse CGH arrays (see E-MEXP-1022). We wanted to validate in our system the known correlation between early replication and expression and to analyze its extent. To this end, we have measured the expression in the same cell line (L1210 cells). Two biological replicates were hybridized to 2 identical microarrays. Expression levels were highly similar between the 2 replicates (r=0.98).
Global organization of replication time zones of the mouse genome.
Cell line, Subject
View SamplesUDP-sugars were identified as extracellular signaling molecules, assigning a new function to these compounds in addition to their well defined role in intracellular substrate metabolism and storage. Previously regarded as an orphan receptor, the G protein-coupled receptor (GPCR) P2Y14 (GPR105) was found to bind extracellular UDP and UDP-sugars. Little is known about the physiological functions of this GPCR. To study its physiological role we used a gene-deficient (KO) mouse strain expressing the bacterial LacZ reporter gene to monitor the physiological expression pattern of P2Y14. We found that P2Y14 is mainly expressed in pancreas and salivary glands and in subpopulations of smooth muscle cells of the gastrointestinal tract, blood vessels, lung and uterus. Among other phenotypical differences KO mice showed a significantly impaired glucose tolerance following oral and intraperitoneal glucose application. An unchanged insulin tolerance suggested altered pancreatic islet function. Transcriptome analysis of pancreatic islets showed that P2Y14 deficiency significantly changed expression of components involved in insulin secretion. Insulin secretion tests revealed a reduced insulin release from P2Y14-deficient islets highlighting P2Y14 as a new modulator of proper insulin secretion. Overall design: 10 samples from pancreatic islets isolated from wildtype mice; 10 samples from pancreatic islets isolated from P2Y14-knockout mice
The G protein-coupled receptor P2Y14 influences insulin release and smooth muscle function in mice.
No sample metadata fields
View SamplesTo compare up-regulation of genes following CpG activation, we performed microarray analysis of activated macrophages from B6 and F1(B6xMOLF) mouse strains. Cells were activated for 0, 2 and 4 hrs with 200nM of type B CpG. Levels of mRNA for many genes differened dramatically between the strains
Mannose receptor 1 mediates cellular uptake and endosomal delivery of CpG-motif containing oligodeoxynucleotides.
Specimen part, Treatment
View SamplesWe used RNA-seq as a method of next generation sequencing (NGS) to identify RIPK1 dependent inflammatory mediators and pathways in LPS injected mice. Overall design: Mice were divided intro 3 groups - control (n=2), LPS (n=2) and LPS/Nec-1 (n=2). BM cells were isolated by FACS as described for qPCR analysis. Total RNAs were isolated using Qiagen RNeasy kit according to the manufacturer's protocol
RIPK1 and RIPK3 Kinases Promote Cell-Death-Independent Inflammation by Toll-like Receptor 4.
Specimen part, Cell line, Treatment, Subject
View SamplesGene level expression estimate using the Whole Transcript (WT) Assay approach of the Gene 1.0 ST Array System for Mouse. This assay was done to identify the RIPK1-dependent gene expression changes in mouse BMDMs.
RIPK1 and RIPK3 Kinases Promote Cell-Death-Independent Inflammation by Toll-like Receptor 4.
Sex, Specimen part
View SamplesSeveral aspects common to a Western lifestyle, including obesity and decreased physical activity, are known risks for gastrointestinal cancers. There is an increasing amount of evidence suggesting that diet profoundly affects the composition of the intestinal microbiota. Moreover, there is now unequivocal evidence linking a dysbiotic gut to cancer development. Yet, the mechanisms through which high-fat diet (HFD)-mediated changes in the microbial community impact the severity of tumorigenesis in the gut, remain to be determined.
High-fat-diet-mediated dysbiosis promotes intestinal carcinogenesis independently of obesity.
Sex, Age, Specimen part, Treatment
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Integrative genomics identifies molecular alterations that challenge the linear model of melanoma progression.
Cell line
View SamplesThe two most common melanoma histopathologic subtypes, superficial spreading (SSM) and nodular melanoma (NM), are believed to represent sequential phases of linear progression from radial to vertical growth. Studies suggest, however, that SSM and NM are biologically distinct. We utilized an integrative genomic approach to examine the possibility that SSM and NM are the result of independent pathways characterized by unique molecular alterations. Cell lines including SSM, NM, metastatic melanoma, and melanocyte controls were evaluated for copy number changes and differential mRNA expression using single nucleotide polymorphism array (SNP 6.0, Affymetrix) and gene array (U133A 2.0, Affymetrix). Data sets were integrated to identify copy number alterations that correlated with gene expression, and array results were validated using immunohistochemistry on human tissue microarrays (TMAs) and an external data set. The functional effect of genomic deletion was assessed by lentiviral overexpression. Integrative genomics revealed 8 genes in which NM/SSM-specific copy number alterations were correlated with NM/SSM differential gene expression (P<0.05, Spearmans rank). Pathways analysis of differentially expressed genes (N=114) showed enrichment for metabolic-related processes. SSM-specific genomic deletions (DIS3, MTAP, G3BP2, SEC23IP, USO1) were verified in an expanded panel of cell lines, and forced overexpression of MTAP in SSM resulted in reduced cell growth. Metabolism-related gene ALDH7A1 was verified as overexpressed in NM using human TMAs.The identification of recurrent genomic deletions in SSM not present in NM challenges the linear model of melanoma progression and supports the unique molecular classification of SSM and NM.
Integrative genomics identifies molecular alterations that challenge the linear model of melanoma progression.
Cell line
View SamplesAlternative mRNA splicing is an important mechanism for regulation of gene expression. Changes in gene expression contribute to the pathogenesis of heart failure. However, changes in mRNA splicing have not been systematically examined in heart disease. We hypothesized that mRNA splicing is changed in diseased hearts.
Heart failure-associated changes in RNA splicing of sarcomere genes.
No sample metadata fields
View SamplesSynthetic DNA-binding proteins have found broad application in gene therapies and as tools for interrogating biology. Engineered proteins based on the CRISPR/Cas9 and TALE systems have been used to alter genomic DNA sequences, control transcription of endogenous genes, and modify epigenetic states. Although the activity of these proteins at their intended genomic target sites have been assessed, the genome-wide effects of their action have not been extensively characterized. Additionally, the role of chromatin structure in determining the binding of CRISPR/Cas9 and TALE proteins to their target sites and the regulation of nearby genes is poorly understood. Characterization of the activity these proteins using modern high-throughput genomic methods would provide valuable insight into the specificity and off-target effects of CRISPR- and TALE-based genome engineering tools. We have analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators targeted to the promoters of two different endogenous human genes in HEK293T cells using a variety of high-throughput DNA sequencing methods. In particular, we assayed the DNA-binding specificity of these proteins and their effects on the epigenome. DNA-binding specificity was evaluated by ChIP-seq and RNA-seq was used to measure the specificity of these activators in perturbing the transcriptome. Additionally, DNase-seq was used to identify the chromatin state at target sites of the synthetic transcriptional activators and the genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these genome engineering technologies are highly specific in both binding to their promoter target sites and inducing expression of downstream genes when multiple activators bind to a single promoter. Moreover, we show that these synthetic activators are able to induce the expression of silent genes in heterochromatic regions of the genome by opening regions of closed chromatin and decreasing DNA methylation. Interestingly, the transcriptional activation domain was not necessary for DNA-binding or chromatin remodeling in these regions, but was critical to inducing gene expression. This study shows that these CRISPR- and TALE-based transcriptional activators are exceptionally specific. Although we detected limited binding of off-target sites in the genome and changes to genome structure, these off-target event did not lead to any detectable changes in gene regulation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. Overall design: HEK293T cells were transfected in triplicate with plasmids expressing synthetic transcription factors. The synthetic TFs were either (a) dCas9-VP64 fusion protein and a targeting guide RNA (gRNA), or (b) a TALE-VP64 fusion protein engineered to bind to a specific target site in the genome. As a control, cells were transfected with plasmids expressing GFP. After transfection, RNA-seq was used to identify both on-target and off-target binding sites for the synthetic TFs. The data in this submission were generated using the TALE transfection experiments.
Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.
No sample metadata fields
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