Project description:Caco-2 cells (a human gastrointestinal epithelial cell line) grown to form confluent cell layers on permeable membrane supports were treated with variously (A) iron depleted medium, (B) iron depleted medium with 10 uM ferric nitrilotiracetate added to the apical medium or (C) iron depleted medium with 30 uM human holo-transferrin added to the basolateral medium for 7 days (days 14-21 after cell seeding). RNA was extract from the cells on day 21 for transcription profiling by array.
Project description:Many signal transduction cascades are initiated by transmembrane receptors with the presence or absence and abundance of receptors dictating cellular responsiveness. We provide a validated array of quantitative reverse transcription polymerase chain reaction (qRT-PCR) reagents for high-throughput profiling of the presence and relative abundance of transcripts for 194 transmembrane receptors in the human genome. We found that the qRT-PCR array had greater sensitivity and specificity for the detected receptor transcript profiles compared to conventional oligonucleotide microarrays or exon microarrays. The qRT-PCR array also distinguished functional receptor presence versus absence more accurately than deep sequencing of adenylated RNA species by RNA sequencing (RNA-seq). By applying qRT-PCR-based receptor transcript profiling to 40 human cell lines representing four main tissues (pancreas, skin, breast, and colon), we identified clusters of cell lines with enhanced signaling capabilities and revealed a role for receptor silencing in defining tissue lineage. Ectopic expression of the interleukin-10 (IL-10) receptor-encoding gene IL10RA in melanoma cells engaged an IL-10 autocrine loop not otherwise present in this cell type, which altered signaling, gene expression, and cellular responses to proinflammatory stimuli. Our array provides a rapid, inexpensive, and convenient means for assigning a receptor signature to any human cell or tissue type.
Project description:ChIP coupled with microarray provides a powerful tool to determine in vivo binding profiling of transcription factors to deduce regulatory circuitries in mammalian cells. Aiming at improving the specificity and sensitivity of such analysis, we developed a new technology called ChIP-DSL using the DNA selection and ligation (DSL) strategy, permitting robust analysis with much reduced materials compared with standard procedures. We profiled general and sequence-specific DNA binding transcription factors using a full human genome promoter array based on the ChIP-DSL technology, revealing an unprecedented number of the estrogen receptor (ERalpha) target genes in MCF-7 cells. Coupled with gene expression profiling, we found that only a fraction of these direct ERalpha target genes were highly responsive to estrogen and that the expression of those ERalpha-bound, estrogen-inducible genes was associated with breast cancer progression in humans. This study demonstrates the power of the ChIP-DSL technology in revealing regulatory gene expression programs that have been previously invisible in the human genome.
Project description:Transcription profiling of antisense transcripts of 10 tissues each from human, mouse, and rat. We profiled the antisense transcription level of 10 tissues each from human, mouse, and rat. Only Affymetrix core probesets were used. Two technical replicates per sample. Reference for protocol: Ge, X., Rubinstein, W.S., Jung, Y.C., and Wu, Q. (2008). Genome-wide analysis of antisense transcription with Affymetrix exon array. BMC Genomics 9, 27.
Project description:We have developed a multianalyte fluid-phase protein array technology termed high-throughput immunophenotyping using transcription (HIT). This method employs a panel of monoclonal antibodies, each tagged with a unique oligonucleotide sequence that serves as a molecular bar code. After staining a sample, T7 polymerase amplifies the tags, which are then hybridized to a DNA microarray for indirect measurement of each analyte. Although there are many potential applications for this technology, here we report its suitability for profiling cytokines, intracellular molecules and cell surface markers. Using HIT, we profiled 90 surface markers on human naive T helper cells activated in vitro. The markers identified in this screen are consistent with previously described activation markers and were validated by flow cytometry. Additionally, a HIT screen of surface markers expressed on T helper cells activated in the presence of transforming growth factor-beta identified downregulation of CD26 in these cells. HIT arrays are an ideal platform for rapidly identifying markers for further characterization and therapeutic intervention.
Project description:1) We identified the genes whose expression was up- and down-regulated by the adhesion to bone marrow stromal cells in human multiple myeloma cell line RPMI8226. 2) We identified the genes whose expression was up- and down-regulated by the PI3K inhibitor PF-04691502 in human multiple myeloma cell line RPMI8226. We isolated mRNA from the multiple myeloma cell line RPMI8226 under drug-resistant conditions, and subjected them to gene expression profiling using an Agilent GeneChip Array.
Project description:The molecular mechanisms responsible for the high immunosuppressive capacity of CD4+ Tregs in tumors are not well known. High-dimensional single-cell profiling of T cells from chemotherapy-naive individuals with non-small-cell lung cancer identified the transcription factor IRF4 as specifically expressed by a subset of intratumoral CD4+ effector Tregs with superior suppressive activity. In contrast to the IRF4- counterparts, IRF4+ Tregs expressed a vast array of suppressive molecules, and their presence correlated with multiple exhausted subpopulations of T cells. Integration of transcriptomic and epigenomic data revealed that IRF4, either alone or in combination with its partner BATF, directly controlled a molecular program responsible for immunosuppression in tumors. Accordingly, deletion of Irf4 exclusively in Tregs resulted in delayed tumor growth in mice while the abundance of IRF4+ Tregs correlated with poor prognosis in patients with multiple human cancers. Thus, a common mechanism underlies immunosuppression in the tumor microenvironment irrespective of the tumor type.
Project description:Individual Streptomyces species have the genetic potential to produce a diverse array of natural products of commercial, medical and veterinary interest. However, these products are often not detectable under laboratory culture conditions. To harness their full biosynthetic potential, it is important to develop a detailed understanding of the regulatory networks that orchestrate their metabolism. Here we integrate nucleotide resolution genome-scale measurements of the transcriptome and translatome of Streptomyces coelicolor, the model antibiotic-producing actinomycete. Our systematic study determines 3,570 transcription start sites and identifies 230 small RNAs and a considerable proportion (?21%) of leaderless mRNAs; this enables deduction of genome-wide promoter architecture. Ribosome profiling reveals that the translation efficiency of secondary metabolic genes is negatively correlated with transcription and that several key antibiotic regulatory genes are translationally induced at transition growth phase. These findings might facilitate the design of new approaches to antibiotic discovery and development.