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SRP070815
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- Drosophila melanogaster
- 3 Downloadable Samples
- Illumina Genome Analyzer IIx, Illumina HiSeq 2000
Description
The discovery of the small regulatory RNA populations has changed our vision of cellular regulations. Indeed, loaded on Argonaute proteins they formed ribonucleoprotein complexes that target complementary sequences and achieved widespread silencing mechanisms conserved in most eukaryotes. The recent development of deep sequencing approaches highly contributed to their detection. Small RNA isolation form cells and/or tissues remains a crucial stage to generate robust and relevant sequencing data. In 2006, a novel strategy based on anion-exchange chromatography has been purposed as an alternative to the standard size-isolation purification procedure. However, the eventual biases of such a method have been poorly investigated. Moreover, this strategy not only relies on advanced technical skills and expensive material but is time consuming and requires an elevated starting biological material amount. Using bioinformatic comparative analysis of six independent small RNA-sequencing libraries of Drosophila ovaries, we here demonstrate that anion-exchange chromatography purification prior to small RNA extraction unbiasedly enriches datasets in bona fide reads (small regulatory RNA reads) and depletes endogenous contaminants (ribosomal RNAs and degradation products). The resulting increase of sequencing depth provides a major benefit to study rare populations. We then developed a fast and basic manual procedure to purify loaded small non coding RNAs using anion-exchange chromatography at the bench. We validated the efficiency of this new method and used this strategy to purify small RNAs from various tissues and organisms. We moreover determined that our manual purification increases the output of the previously described anion-exchange chromatography procedure. Overall design: Comparison of small regulatory RNA populations obtained after three different small RNA purification procedures
Publication Title
A user-friendly chromatographic method to purify small regulatory RNAs.
Sample Metadata Fields
Sex, Specimen part, Cell line, Subject
View Samples- Drosophila melanogaster
- 6 Downloadable Samples
- Illumina HiSeq 2000, Illumina Genome Analyzer II
Description
The maintenance of genome integrity is an essential trait to the successful transmission of genetic information. In animal germ cells, piRNAs guide PIWI proteins to silence transposable elements (TEs) in order to maintain genome integrity. In insects, most of TE silencing in the germline is achieved by secondary piRNAs that are produced by a feed-forward loop (the ping-pong cycle), which requires the piRNA-directed cleavages of two types of RNAs: mRNAs of functional euchromatic TEs and heterochromatic transcripts that contain defective TE sequences. The first cleavage which initiates such amplification loop remains poorly understood. Taking advantage of the existence of strains that are devoid of functional copies of the LINE-like I-element, we report that in such Drosophila ovaries, the initiation of a ping-pong cycle is achieved only by secondary I-element piRNAs that are produced in the ovary and deposited in the embryonic germline. This unusual secondary piRNA biogenesis, detected in the absence of functional I-element copies, results from the processing of sense and antisense transcripts of several different defective I-elements. Once acquired, for instance after ancestor aging, this capacity to produce heterochromatic-only secondary piRNAs is partially transmitted through generations via maternal piRNAs. Furthermore, such piRNAs acting as ping-pong initiators in a chromatin-independent manner confer to the progeny a high capacity to repress the I-element mobility. Our study explains at the molecular level the basis for epigenetic memory of maternal immunity that protects females from hybrid dysgenesis caused by transposition of paternally inherited functional I-elements. Overall design: Comparison of Drosophila small RNA populations in ovaries and/or eggs from 3-day-old or 25-day-old females.
Publication Title
piRNA-mediated transgenerational inheritance of an acquired trait.
Sample Metadata Fields
Sex, Age, Specimen part, Cell line, Subject
View Samples- Homo sapiens
- 52 Downloadable Samples
Affymetrix HT Human Genome U133A Array (hthgu133a)
Description
We have generated a large collection of normal human mammary epithelial cell strains from women aged 16 to 91 years, derived from primary tissues, to enable functional and molecular interrogation of aging. We demonstrate in finite-lifespan cultured and uncultured epithelial cells that aging is associated with reduction of myoepithelial cells and with increases in luminal cells expressing keratin 14 and integrin 6, traits that are expressed exclusively in myoepithelial cells in women under 30. We find that changes to the luminal lineage result from age-dependent expansion of multipotent progenitors that bear defects resulting in incompletely differentiated luminal cells. These findings were verified in vivo in normal breast tissues. Myoepithelial cells have been suggested to act as tumor suppressors, and progenitor cells are implicated as the etiological roots of mammary carcinomas. Thus with aging there is a shift in the balance of luminal/myoepithelial lineages, and changes in the functional spectrum of multipotent progenitors, which presages increased potential for malignant transformation.
Publication Title
Accumulation of multipotent progenitors with a basal differentiation bias during aging of human mammary epithelia.
Sample Metadata Fields
Age, Specimen part
View Samples- Mus musculus
- 8 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
The overall goal of this project is to investigate the role of TGF-beta signaling in tissue-tissue interactions between myogenic precursors of craniofacial muscles and cranial neural crest cells (CNCCs). Here, we conducted gene expression profiling of the tongue bud from mice at embryonic day E13.5 with a CNCC-specific conditional inactivation of the TGF-beta receptor type 1 gene Alk5. These mice provide a model of microglossia as well as disrupted extraocular and masticatory muscle development, which are congenital birth defects commonly observed in several syndromic conditions.
Publication Title
ALK5-mediated transforming growth factor β signaling in neural crest cells controls craniofacial muscle development via tissue-tissue interactions.
Sample Metadata Fields
Sex, Specimen part
View Samples- Mus musculus
- 8 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
The overall goal of this project is to investigate the role of TGF-beta signaling in tissue-tissue interactions between myogenic precursors of craniofacial muscles and cranial neural crest cells (CNCCs). Here, we conducted gene expression profiling of the mandibular arch from mice at embryonic day E11.5 with a CNCC-specific conditional inactivation of the TGF-beta receptor type 1 gene Alk5. These mice provide a model of microglossia as well as disrupted extraocular and masticatory muscle development, which are congenital birth defects commonly observed in several syndromic conditions.
Publication Title
ALK5-mediated transforming growth factor β signaling in neural crest cells controls craniofacial muscle development via tissue-tissue interactions.
Sample Metadata Fields
Sex, Specimen part
View Samples- Mus musculus
- 6 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
The overall goal of this project is to investigate the role of TGF-beta signaling in tongue development in order to study the contribution of cranial neural crest (CNC) cells towards the patterning of cranial mesoderm for proper tongue formation. Here, we conducted gene expression profiling of embryonic tongue tissue from wild type mice as well as those with a neural crest specific conditional inactivation of the Tgfbr2 gene. The latter mice provide a model of microglossia, a common congenital birth defect which is frequently observed with several syndromic conditions.
Publication Title
Noncanonical transforming growth factor β (TGFβ) signaling in cranial neural crest cells causes tongue muscle developmental defects.
Sample Metadata Fields
Sex, Specimen part
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