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GSE37271
Homo sapiens
18 Downloadable Samples
Affymetrix Human Gene 1.0 ST Array (hugene10st)
Description
This study is to find the cellular and molecular mechanisms by which a naturally-occurring Np53 isoform causes accelerated aging in humans. The biological function of Np53, which lacks only 40 N-terminal amino acids, represents an example of p53 as a regulator of mammalian aging. When expressed together with WTp53 in mice, Np53 causes an aging phenotype such as shorter life span, reduced body mass, organ atrophy and osteoporosis. Because p53 must form a tetramer to regulate transcription, we generated p53 clones (based upon the structure of the native p53 tetramer) containing one Np53 linked with one WTp53 to form a functional Np53:WTp53 tetramer with 1:1 stoichiometry. Thus, our strategy ensured each p53 tetramer contained 2 Np53 and 2 WTp53 proteins. Importantly, Np53:WTp53 form stable tetramers, based upon gel filtration chromatography and structural analysis using electron microscopy. Furthermore, the Np53:WTp53 tetramer activates transcription equally well compared with WTp53 tetramers in an in vitro reconstituted transcription system. Having verified the stoichiometry, stability, structure, and activity of these Np53:WTp53 tetramers, here we used microarray analysis to compare global gene expression patterns in p53-null H1299 cells expressing either WTp53 or Np53:WTp53. As expected, global gene expression was largely similar, since the differences between Np53:WTp53 tetramers and WTp53 tetramers are slight: only 2 of 4 p53 proteins will be different in the Np53:WTp53 tetramer. Among only several dozen genes that were selectively up- or down-regulated 2-fold or greater, many genes known to regulate mammalian aging were altered in cells expressing Np53:WTp53, including insulin signaling pathway members (IRS1, INPP5D, PLK3, MAP3K1, FGF5) and regulators of glucose metabolism (SLC2A2, CRYAB, LRCH1). Expression of other key metabolic genes were also altered in cells expressing Np53:WTp53 tetramers, suggesting that global me tabolic changes might contribute to Np53:WTp53 pathology. In collaboration with Metabolon (Durham, NC), we identified approximately one hundred metabolites that were significantly up- or down-regulated in H1299 cells expressing Np53:WTp53. The metabolome analysis was a powerful complement to the gene expression data, and further suggested that the mTOR pathway (e.g. across-the-board up-regulation of amino acid levels) and mitochondrial function (e.g. up-regulation of carnitine, important for a-oxidation of fatty acids) was altered in cells expressing Np53:WTp53. These findings were subsequently validated using biochemical and cell-based approaches. Furthermore, whereas equal expression of Np53 and WTp53 cause accelerated aging in mammals, due to alternative splicing and translation initiation Np53 is a naturally-occurring isoform whose expression levels can change throughout the lifetime. Thus, the cellular and molecular mechanisms identified from this work will likely reflect changes common to normal, physiological aging.
Publication Title
The human ΔNp53 isoform triggers metabolic and gene expression changes that activate mTOR and alter mitochondrial function.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Homo sapiens
- 8 Downloadable Samples
- Affymetrix Human Gene 1.0 ST Array (hugene10st)
Description
The Mediator complex allows communication between transcription factors and RNA polymerase II (RNAPII). CDK8, the kinase found in some variants of Mediator, has been characterized mostly as a transcriptional repressor. Recently, CDK8 was demonstrated to be a potent oncoprotein. Here we show that CDK8 is predominantly a positive regulator of gene expression within the serum response network, as it is required for expression of several members of the AP-1 and EGR family of oncogenic transcription factors (e.g. FOS, JUN, EGR1-3). Mechanistic studies demonstrate that CDK8 is not required for recruitment of RNAPII and promoter escape at these loci. Instead, CDK8 depletion leads to the appearance of slower elongation complexes carrying hypophosphorylated RNAPII. We show that CDK8-Mediator regulates precise steps in the assembly of a functional elongation complex, including the recruitment of P-TEFb and BRD4, but is dispensable for recruitment of SPT5 and FACT. Furthermore, CDK8-Mediator specifically interacts with P-TEFb. Thus, we uncovered a novel role for CDK8 in transcriptional regulation that may contribute to its oncogenic effects.
Publication Title
CDK8 is a positive regulator of transcriptional elongation within the serum response network.
Sample Metadata Fields
Cell line
View Samples- Homo sapiens
- 6 Downloadable Samples
IlluminaHiSeq2500
Description
Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we report here the first large-scale identification of Mediator kinase substrates in human cells (HCT116). Among over 16,000 quantified phosphosites, we identified 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-Seq data correlated with Mediator kinase targets, CA effects on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, which tracked about 7,000 proteins across six time points (0 – 24h), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation; contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest cellular roles beyond transcription, including metabolism and DNA repair. Overall design: HCT116 cells were treated with either 100nM CA or DMSO in biological triplicate for each population (6 samples total). Treatment was for 24h for compound and vehicle.
Publication Title
Identification of Mediator Kinase Substrates in Human Cells using Cortistatin A and Quantitative Phosphoproteomics.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 8 Downloadable Samples
- Illumina HiSeq 2500
Description
Gene expression is controlled by transcription factors (TFs) that consist of DNA-binding domains (DBDs) and activation domains (ADs). The DBDs have been well- characterized, but little is known about the mechanisms by which ADs effect gene activation. Here we report that diverse ADs form phase-separated condensates with the Mediator coactivator. For the OCT4 and GCN4 TFs, we show that the ability to form phase-separated droplets with Mediator in vitro and the ability to activate genes in vivo are dependent on the same amino acid residues. For the estrogen receptor (ER), a ligand-dependent activator, we show that estrogen enhances phase separation with Mediator, again linking phase separation with gene activation. These results suggest that diverse TFs can interact with Mediator through the phase-separating capacity of their ADs and that formation of condensates with Mediator is involved in gene activation. Overall design: RNA-seq in mouse embryonic stem cells after OCT4 degradation or LIF withdrawal
Publication Title
Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains.
Sample Metadata Fields
Treatment, Subject
View Samples- Homo sapiens
- 24 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA, IRF8, IRF1 and ETV6. The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.
Publication Title
Mediator kinase inhibition further activates super-enhancer-associated genes in AML.
Sample Metadata Fields
Specimen part, Cell line, Treatment
View Samples- Homo sapiens
- 6 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
We characterized the marine natural product cortistatin A (CA) as an inhibitor of CDK8 to determine whether pharmacologic inhibition of CDK8 regulates super-enhancer function and inhibits AML proliferation.
Publication Title
Mediator kinase inhibition further activates super-enhancer-associated genes in AML.
Sample Metadata Fields
Specimen part, Cell line, Treatment
View Samples- Homo sapiens
- 6 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
We characterized the marine natural product cortistatin A (CA) as an inhibitor of CDK8 to determine whether pharmacologic inhibition of CDK8 regulates super-enhancer function and inhibits AML proliferation.
Publication Title
Mediator kinase inhibition further activates super-enhancer-associated genes in AML.
Sample Metadata Fields
Specimen part, Cell line, Treatment
View Samples- Homo sapiens
- 6 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
We characterized the marine natural product cortistatin A (CA) as an inhibitor of CDK8 to determine whether pharmacologic inhibition of CDK8 regulates super-enhancer function and inhibits AML proliferation.
Publication Title
Mediator kinase inhibition further activates super-enhancer-associated genes in AML.
Sample Metadata Fields
Specimen part, Cell line, Treatment
View Samples- Homo sapiens
- 6 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
We characterized the marine natural product cortistatin A (CA) as an inhibitor of CDK8 to determine whether pharmacologic inhibition of CDK8 regulates super-enhancer function and inhibits AML proliferation.
Publication Title
Mediator kinase inhibition further activates super-enhancer-associated genes in AML.
Sample Metadata Fields
Specimen part, Cell line, Treatment
View Samples- Homo sapiens
- 6 Downloadable Samples
- Illumina HiSeq 2000
Description
We characterized the marine natural product cortistatin A (CA) as an inhibitor of CDK8 to determine whether pharmacologic inhibition of CDK8 regulates super-enhancer function and inhibits AML proliferation. In this series, we examine the transcriptional effect on insensitive HCT116 cells of 3hrs exposure to CA. Overall design: HCT116 cells were treated in triplicate with either DMSO or CA for 3hrs after which RNA was harvested and prepared for RNA sequencing to assess transcriptional changes.
Publication Title
Mediator kinase inhibition further activates super-enhancer-associated genes in AML.
Sample Metadata Fields
No sample metadata fields
View Samples