The organization of mammalian DNA replication is poorly understood. We have produced genome-wide high-resolution dynamic maps of the timing of replication in human erythroid, mesenchymal and embryonic stem cells using TimEX, a method that relies on gaussian convolution of massive, highly redundant determinations of DNA copy number variations during S phase obtained using either high-density oligonucleotide tiling arrays or massively-parallel sequencing to produce replication timing profiles. We show that in untransformed human cells, timing of replication is highly regulated and highly synchronous, and that many genomic segments are replicated in temporal transition regions devoid of initiation where replication forks progress unidirectionally from origins that can be hundreds of kilobases away. Absence of initiation in one transition region is shown at the molecular level by SMARD analysis. Comparison of ES and erythroid cells replication patterns revealed that these cells replicate about 20% of their genome in different quarter of S phase and that ES cells replicate a larger proportion of their genome in early S phase than erythroid cells. Importantly, we detected a strong inverse relationship between timing of replication and distance to the closest expressed gene. This relationship can be used to predict tissue specific timing of replication profiles from expression data and genomic annotations. We also provide evidence that early origins of replication are preferentially located near highly expressed genes, that mid firing origins are located near moderately expressed genes and that late firing origins are located far from genes.
Predictable dynamic program of timing of DNA replication in human cells.
Specimen part
View SamplesThe comparative advantages of RNA-Seq and microarrays in transcriptome profiling were evaluated in the context of a comprehensive study design. Gene expression data from Illumina RNA-Seq and Affymetrix microarrays were obtained from livers of rats exposed to 27 agents that comprised of seven modes of action (MOAs); they were split into training and test sets and verified with real time PCR. Overall design: 105 samples were selected from the DrugMatirx tissue/RNA bank that is now owned by the National Toxicology Program (NTP). The samples were split into 2 sets, training and test, to allow for the evaluation of classifiers derived from the data. There were 63 samples in the training set and 42 in the test set. Of the 63 samples in the training set 45 were derived from rats treated with test agent and 18 were control samples (3 sets of 6). 39 of the test set samples were derived from test agent treated animals and 6 were from vehicle and route matched controls. Five MOAs were represented in the training set and 4 MOAs were in the test set. Two of the MOAs were duplicated from the test set and two were without representation in the training set. For each test agent there were three rats treated, in accordance with the common practice in the field of toxicology. For each MOA there were three representative test agents to ensure adequate power for detecting the MOA signatures. 6 samples from the training set had duplicate libraries sequenced and duplicate sequencing runs for the first library. DrugMatrix, National Toxicology program (NTP) Sequencing was carried out in Dr. Charles Wang's Functional Genomics Core at City of Hope Comprehensive Cancer Center, Duarte, CA
Transcriptomic profiling of rat liver samples in a comprehensive study design by RNA-Seq.
No sample metadata fields
View SamplesInterleukin-21 (IL-21) is a pleiotropic cytokine that induces expression of transcription factor BLIMP1 (encoded by Prdm1), which regulates plasma cell differentiation and T cell homeostasis. We identified an IL-21 response element downstream of Prdm1 that binds the transcription factors STAT3 and IRF4, which are required for optimal Prdm1 expression. Genome-wide ChIP-Seq mapping of STAT3- and IRF4-binding sites showed that most regions with IL-21-induced STAT3 binding also bound IRF4 in vivo, and furthermore, revealed that the noncanonical TTCnnnTAA GAS motif critical in Prdm1 was broadly used for STAT3 binding. Comparing genome-wide expression array data to binding sites revealed that most IL-21-regulated genes were associated with combined STAT3-IRF4 sites rather than pure STAT3 sites. Correspondingly, ChIP-Seq analysis of Irf4_/_ T cells showed greatly diminished STAT3 binding after IL-21 treatment, and Irf4_/_ mice showed impaired IL- 21-induced Tfh cell differentiation in vivo. These results reveal broad cooperative gene regulation by STAT3 and IRF4.
Analysis of interleukin-21-induced Prdm1 gene regulation reveals functional cooperation of STAT3 and IRF4 transcription factors.
Specimen part
View SamplesThe comparative advantages of RNA-Seq and microarrays in transcriptome profiling were evaluated in the context of a comprehensive study design. Gene expression data from Illumina RNA-Seq and Affymetrix microarrays were obtained from livers of rats exposed to 27 agents that comprised of seven modes of action (MOAs); they were split into training and test sets and verified with real time PCR.
The concordance between RNA-seq and microarray data depends on chemical treatment and transcript abundance.
Sex, Specimen part
View SamplesDifferential gene expression was analyzed for FACS sorted Math1::Cre; ROSA-tdTomato from hand dissected cochlear nuclei of wild type and Hoxa2/Hoxb2 mutant mice Overall design: In order to investigate the role of Hoxa2 and Hoxb2 transcription factors in a subset of cells of the cochlear nucleus, we generated double conditional knock-out by crossing the deleter line Math1::Cre crossed with Rosa tdTomato; Hoxa2fl/fl; Hoxb2fl/fl and Rosa tdTomato wild type background. FACS sorted cells from hand dissected cochlear nuclei were than processed and RNA-seq performed (see extract protocol and library construction protocol).
Hox2 Genes Are Required for Tonotopic Map Precision and Sound Discrimination in the Mouse Auditory Brainstem.
No sample metadata fields
View SamplesNeuroprotective therapies for retinal degeneration may be used to rescue retinal cells and preserve vision. Hypoxic preconditioning stabilizes the transcription factor HIF-1 in the retina and strongly protects photoreceptors in an animal model of light-induced retinal degeneration.
Analysis of the retinal gene expression profile after hypoxic preconditioning identifies candidate genes for neuroprotection.
No sample metadata fields
View SamplesMicroarray technology has had a profound impact on gene expression research. Some studies have questioned whether similar expression results are obtained when the same RNA samples are analyzed on different platforms.
The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements.
No sample metadata fields
View SamplesSignal transduction processes mediated by phosphatidyl inositol phosphates affect a broad range of cellular processes such as cell cycle progression, migration and cell survival. The protein kinase AKT is one of the major effectors in this signaling network. Chronic AKT activation contributes to oncogenic transformation and tumor development. Therefore, new small drugs were designed to block AKT activity for cancer treatment.
Characterization of AKT independent effects of the synthetic AKT inhibitors SH-5 and SH-6 using an integrated approach combining transcriptomic profiling and signaling pathway perturbations.
Specimen part, Cell line
View SamplesChemo-resistance to platinum such as cisplatin is critical in the treatment of ovarian cancer. Recent evidences have linked epithelial-mesenchymal transition (EMT) with the drug resistance as a contributing mechanism. The current study explored the connection between cellular responses to cisplatin with EMT in ovarian cancer.
Epithelial-mesenchymal status renders differential responses to cisplatin in ovarian cancer.
Specimen part, Cell line, Treatment
View SamplesFor more than a decade, microarrays have been a powerful and widely used tool to explore the transcriptome of biological systems. However, the amount of biological material from cell sorting or laser capture microdissection is much too small to perform microarray studies. To address this issue, RNA amplification methods have been developed to generate sufficient targets from picogram amounts of total RNA to perform microarray hybridisation. In this study, four commercial protocols for amplification of picograms amounts of input RNA for microarray expression profiling were evaluated and compared. The quantitative and qualitative performances of the methods were assessed. Microarrays were hybridised with the amplified targets and the amplification protocols were compared with respect to the quality of expression profiles, reproducibility within a concentration range of input RNA, and sensitivity.
Evaluation of methods for amplification of picogram amounts of total RNA for whole genome expression profiling.
No sample metadata fields
View Samples