By using high-density DNA microarrays, we analyzed the gene-expression profile of SHSY5Y neuroblastoma cells after treatment with cobalt chloride
Investigation of Endogenous Retrovirus Sequences in the Neighborhood of Genes Up-regulated in a Neuroblastoma Model after Treatment with Hypoxia-Mimetic Cobalt Chloride.
Specimen part, Cell line
View SamplesArgonaute (Ago) proteins, which act in post-transcriptional gene regulation directed by small RNAs, are vital for normal stem cell biology. Here we report the genomic characterization of stable Ago-deficient mouse embryonic stem cells (mESC) and determine the direct, primary and system level response to loss of Ago-mediated regulation. We find mESCs lacking all four Ago proteins are viable, do not repress microRNA (miRNA)-targeted cellular RNAs, and show distinctive gene network signatures. Profiling of RNA expression and epigenetic activity in an Ago mutant genetic series indicates that early responses to Ago loss are driven by transcriptional regulatory networks, in particular the Tgf-ß/Smad transcriptional network. This finding is confirmed using a time course analysis of Ago depletion and Ago rescue experiments. Detailed analysis places Tgf-ß/Smad activation upstream of cell cycle regulator activation, such as Cdkn1a, and repression of the c-Myc transcriptional network. The Tgf-ß/Smad pathway is directly controlled by multiple low-affinity miRNA interactions with Tgf-ß/Activin receptor mRNAs and receptor-mediated activation is required for Tgf-ß/Smad target induction with Ago loss. Our characterization reveals the interplay of post-transcriptional regulatory pathways with transcriptional networks in maintaining cell state and likely coordinating cell state transitions. Overall design: mRNA seq from stable genetic Dicer and Dgcr8 mutant mouse embryonic stem cells.
Temporal Control of the TGF-β Signaling Network by Mouse ESC MicroRNA Targets of Different Affinities.
Specimen part, Cell line, Subject
View SamplesPrimary human macrophages with a HIF-1alpha or HIF-2alpha knockdown were pretreated with IL-10 for 16h and afterwards for 4h additionaly under hypoxi (1% O2), RNA was isolated usind the Qiagen RNAeasy Kit and cDNA synthesis wos done using Ambion WT Expression Kit. Expression was compared to si control under control conditions.
Genome-wide identification of hypoxia-inducible factor-1 and -2 binding sites in hypoxic human macrophages alternatively activated by IL-10.
Specimen part
View SamplesThe goal of the experiment was to assay the role of the glucocorticoid receptor (GR) in development of mesenchynmal cells of the lung occuring between the 16 and 18 day of embryonal development.
Glucocorticoid activity during lung maturation is essential in mesenchymal and less in alveolar epithelial cells.
Specimen part
View SamplesAnalysis of newborn mouse epidermis lacking the expression of Insulin receptor (IR) and Insulin like growth factor 1 receptor (IGF-1R). Results show that IR/IGF-1R signalling control epidermal morphogenesis.
Insulin/IGF-1 controls epidermal morphogenesis via regulation of FoxO-mediated p63 inhibition.
No sample metadata fields
View SamplesPromoter-proximal pausing of RNA polymerase II (Pol II) is a widespread transcriptional regulatory step across metazoans. Here we find that the nuclear exon junction complex (pre-EJC) is a critical and conserved regulator of this process. Depletion of pre-EJC subunits leads to a global decrease in Pol II pausing and to premature entry into elongation. This effect occurs, at least in part, via non-canonical recruitment of pre-EJC components at promoters. Failure to recruit the pre-EJC at promoters results in increased binding of the positive transcription elongation complex (P-TEFb) and in enhanced Pol II release. Notably, restoring pausing is sufficient to rescue exon skipping and the photoreceptor differentiation defect associated with depletion of pre-EJC components in vivo. We propose that the pre-EJC serves as an early transcriptional checkpoint to prevent premature entry into elongation, ensuring proper recruitment of RNA processing components that are necessary for exon definition. Overall design: polyA mRNA -seq in conditions with the indicated knockdown treatments
Promoter-proximal pausing mediated by the exon junction complex regulates splicing.
Specimen part, Cell line, Subject
View Samples10 days old tumor spheroids were processed for RNA isolation using the Quiagen RNeasy Micro Kit and cDNA synthesis was done using the Ambion WT Expression Kit. Wt, HIF-1 k/d and HIF-2 k/d samples were compared to each other.
HIF-2alpha-dependent PAI-1 induction contributes to angiogenesis in hepatocellular carcinoma.
Specimen part, Cell line
View SamplesArabidopsis etiolated seedlings (4d old) Col-0 wild type compared to det3 mutants under various growth conditions
Reduced V-ATPase activity in the trans-Golgi network causes oxylipin-dependent hypocotyl growth Inhibition in Arabidopsis.
Age
View SamplesIdentifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs. Overall design: Two replicates of three cDNA libraries were submitted to deep sequencing: a sample from RNA-7-transfected cells; a sample from pre-miR-106a transfected cells; and a control sample.
miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction.
No sample metadata fields
View SamplesIdentifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs. Overall design: Two replicates of two cDNA libraries were submitted to deep sequencing: a sample from siH19-transfected cells and a control sample.
miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction.
No sample metadata fields
View Samples