Project description:Interferons (IFNs) are key players in the antiviral response. IFN sensing by the cell activates transcription of IFN-stimulated genes (ISGs) able to induce an antiviral state by affecting viral replication and release. IFN also induces the expression of ISGs that function as negative regulators to limit the strength and duration of IFN response. The ISGs identified so far belong to coding genes. However, only a small proportion of the transcriptome corresponds to coding transcripts and it has been estimated that there could be as many coding as long non-coding RNAs (lncRNAs). To address whether IFN can also regulate the expression of lncRNAs, we analyzed the transcriptome of HuH7 cells treated or not with IFNα2 by expression arrays. Analysis of the arrays showed increased levels of several well-characterized coding genes that respond to IFN both at early or late times. Furthermore, we identified several IFN-stimulated or -downregulated lncRNAs (ISRs and IDRs). Further validation showed that ISR2, 8, and 12 expression mimics that of their neighboring genes GBP1, IRF1, and IL6, respectively, all related to the IFN response. These genes are induced in response to different doses of IFNα2 in different cell lines at early (ISR2 or 8) or later (ISR12) time points. IFNβ also induced the expression of these lncRNAs. ISR2 and 8 were also induced by an influenza virus unable to block the IFN response but not by other wild-type lytic viruses tested. Surprisingly, both ISR2 and 8 were significantly upregulated in cultured cells and livers from patients infected with HCV. Increased levels of ISR2 were also detected in patients chronically infected with HIV. This is relevant as genome-wide guilt-by-association studies predict that ISR2, 8, and 12 may function in viral processes, in the IFN pathway and the antiviral response. Therefore, we propose that these lncRNAs could be induced by IFN to function as positive or negative regulators of the antiviral response. HuH7 cells were treated with 10000 units/ml of IFN a2 and RNA was isolated 3 days post-treatment
Project description:It has been recently shown that the transcription factor p53 induces the expression of multiple lincRNAs. However, relatively little is known about the role that lincRNAs play in this pathway. Here we characterize a lincRNA named PINT (p53 Induced Noncoding Transcript). We show that PINT is a ubiquitously expressed lincRNA that is finely regulated by p53. In mouse cells, PINT promotes cell proliferation and survival by regulating the expression of genes of TGF-beta, MAPK and p53 pathways. PINT is a nuclear lincRNA that directly interacts with Polycomb Repressive Complex 2 (PRC2), being required for PRC2 targeting of specific genes for repression. Furthermore, PINT functional activity is dependent on PRC2 expression, representing a connection between the p53 pathway and epigenetic regulation by PRC2. We have also identified PINT human ortholog (hPINT), which presents suggestive analogies with the mouse lincRNA. hPINT is similarly regulated by p53, and its expression correlates significantly with the same cellular pathways as the mouse ortholog, including the p53 pathway. Interestingly, hPINT is significantly downregulated in colon cancer, representing a novel tumor suppressor candidate. Our results not only help our understanding of the role of p53 and lincRNAs in cancer, but also contribute to the open debate regarding the utility of mouse models for the study of lincRNAs. Normal tissue located at least 20 cm away from a tumor was obtained through surgical resection from patients of colorectal cancer. Total RNA was extracted and subjected to microarray analysis.
Project description:Interferons (IFNs) are key players in the antiviral response. IFN sensing by the cell activates transcription of IFN-stimulated genes (ISGs) able to induce an antiviral state by affecting viral replication and release. IFN also induces the expression of ISGs that function as negative regulators to limit the strength and duration of IFN response. The ISGs identified so far belong to coding genes. However, only a small proportion of the transcriptome corresponds to coding transcripts and it has been estimated that there could be as many coding as long non-coding RNAs (lncRNAs). To address whether IFN can also regulate the expression of lncRNAs, we analyzed the transcriptome of HuH7 cells treated or not with IFNα2 by expression arrays. Analysis of the arrays showed increased levels of several well-characterized coding genes that respond to IFN both at early or late times. Furthermore, we identified several IFN-stimulated or -downregulated lncRNAs (ISRs and IDRs). Further validation showed that ISR2, 8, and 12 expression mimics that of their neighboring genes GBP1, IRF1, and IL6, respectively, all related to the IFN response. These genes are induced in response to different doses of IFNα2 in different cell lines at early (ISR2 or 8) or later (ISR12) time points. IFNβ also induced the expression of these lncRNAs. ISR2 and 8 were also induced by an influenza virus unable to block the IFN response but not by other wild-type lytic viruses tested. Surprisingly, both ISR2 and 8 were significantly upregulated in cultured cells and livers from patients infected with HCV. Increased levels of ISR2 were also detected in patients chronically infected with HIV. This is relevant as genome-wide guilt-by-association studies predict that ISR2, 8, and 12 may function in viral processes, in the IFN pathway and the antiviral response. Therefore, we propose that these lncRNAs could be induced by IFN to function as positive or negative regulators of the antiviral response. HuH7 cells were treated with 10000 units/ml of IFN a2 and RNA was isolated 3 days post-treatment
Project description:Neurotrophic factors (NTFs) are a relevant group of secreted proteins that modulate growth, differentiation, repair, and survival of neurons; playing a role in the maintenance of the synaptic unions, dendrites and axons; also, being crucial for peripheral nervous system development and regulating plasticity in the adult central nervous system. On the other hand, insulin-like growth factor 1 (IGF-1) has been ascertained multiple beneficial actions in the brain: neuro-development, -protection, -genesis and plasticity. To further investigate the possible mechanisms underlying IGF-1 deficiency in the establishment of neurological disease, microarray and RT-qPCR gene expression analyses coupled with in silico processing were performed in an experimental model of partial IGF-1 deficiency. Results show that the mere IGF-1 deficiency seems to be responsible for an altered expression of genes coding for neurotrophic factors (particularly ciliary neurotrophic factor and mesencephalic astrocyte-derived neurotrophic factor), their receptors and signaling pathways (specially RET). The presented findings support that IGF-1 deficiency might be involved in the establishment and progression of neurodegenerative disorders.
Project description:High-throughput RNA sequencing (RNA-Seq) has enabled accurate gene discovery and expression estimation, but robust differential analysis of gene and transcript abundances has proven difficult. We present a new algorithm, implemented in the freely available tool Cuffdiff, which integrates transcript-level expression estimation with a method to control for variability across replicate samples. Cuffdiff robustly identifies differentially regulated isoforms and genes and reveals differential splicing or promoter-preference changes. We demonstrate the accuracy of our approach through differential analysis of lung fibroblasts in response to loss of the developmental transcription factor HOXA1 and uncover a critical role for this gene in the maintenance of adult cells. We show that HOXA1 is required for lung fibroblast and HeLa cell cycle progression, and loss of HOXA1 results in significant expression level changes in thousands of individual transcripts, along with isoform switching events in key regulators of the cell cycle. Lung Fibroblasts were transfected with either a HOXA1 directed siRNA pool or a scramble non-targeting siRNA control. RNA was collected 48 hours after transfection and changes in gene expression were assayed for using Agilent microarrays.
Project description:Earlier work demonstrated that the transcription factor C/EBPα can convert immature and mature murine B lineage cells into functional macrophages. Testing >20 human lymphoma and leukemia B-cell lines, we found that most can be transdifferentiated at least partially into macrophage-like cells, provided that C/EBPα is expressed at sufficiently high levels. A tamoxifen-inducible subclone of the Seraphina Burkitt lymphoma line, expressing C/EBPαER, could be efficiently converted into phagocytic and quiescent cells with a transcriptome resembling normal macrophages. The converted cells retained their phenotype even when C/EBPα was inactivated, a hallmark of cell reprogramming. Interestingly, C/EBPα induction also impaired the cells' tumorigenicity. Likewise, C/EBPα efficiently converted a B- lymphoblastic leukemia cell line into macrophage-like cells, again dramatically impairing their tumorigenicity. Our experiments show that human cancer cells can be induced to transdifferentiate by C/EBPα into seemingly normal cells at high frequencies and provide a proof of principle for a potential new therapeutic strategy to treat B-cell malignancies. Changes in gene expression during transdifferentiation of BLaER1 cells, comparing uninduced (0h) cells with cells treated with E2 for 3h, 6h, 9h, 12h, 18h, 24h, 36h, 48h, 72h, 120h or 168h. Primary human B-cells and macrophages were used as controls. 2 replicates each.
Project description:This SuperSeries is composed of the following subset Series: GSE30038: Programming human pluripotent stem cells into adipocytes [Affymetrix] GSE30039: Programming human pluripotent stem cells into adipocytes [Agilent] Refer to individual Series
Project description:The utility of human pluripotent stem cells as a tool for understanding disease and as a renewable source of cells for transplantation therapies is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into adipocytes. We found that inducible expression of PPARG2 in pluripotent stem cell-derived mesenchymal progenitor cells programmed their development towards an adipocyte cell fate. Using this approach, multiple human pluripotent cell lines were differentiated into adipocytes with efficiencies of 85% to 90%. These pluripotent stem cell-derived adipocytes retained their identity independent of transgene expression, could be maintained in culture for several weeks, expressed mature markers, and exhibited mature functional properties such as lipid catabolism in response to a beta-adrenergic stimulus. Global transcriptional and lipid metabolomic analyses further confirmed the identity and maturity of these pluripotent stem cell-derived adipocytes. Mesenchymal progenitor cells (MPCs) derived from human embryonic stem cells hESCs and induced pluripotent stem cells (iPSCs) along with adipose-derived stromal vascular cells (ADSVCs) were subjected to induction of PPAR2 and compared to primary fat samples. Overall 2 ADSVC (ADSVC 24 nd 49) lines, 1 hESC (HUES9) line and 1 iPSC (BJRiPS) line were differentiated into MPCs, PPAR2 programmed, and compared to untreated MPCs and primary fat samples from 2 individuals. Each condition is either represented in duplicate or triplicate and there are two universal reference spots to aid in slide-dependant batch effects (24 samples total). Supplementary file(s): GeneSymbol-collapsed data represent the final normalized data used for analyses in the manuscript.