Transcription profiling by array of Dictyostelium discoideum cdk8- cells and parental cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Dictyostelium discoideum
SUBMITTER: Gareth Bloomfield
PROVIDER: E-TABM-1013 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We report here that duplications of 15 kb or more are common in the genome of the social amoeba Dictyostelium discoideum. Most of the axenic "workhorse" strains Ax2 and Ax3/4 obtained from different laboratories can be expected to carry new duplications. The auxotrophic strains DH1 and JH10 also bear previously unreported duplications. Strain Ax3/4 is known to carry a large duplication on chromosome 2 and the domain boundary of this structure shows evidence of further instability; we find a further variable duplication on chromosome 5. These duplications are lacking in Ax2, which has instead a small duplication on chromosome 1. Stocks of the type isolate NC4 are similarly variable, though we have identified some approximating the assumed ancestral genotype. More recent wild-type isolates are almost without duplications, but we can identify small deletions or regions of high divergence, possibly reflecting responses to local selective pressures. Duplications are scattered through most of the genome, and can be stable enough to reconstruct genealogies spanning decades of the history of the NC4 lineage. The expression level of many duplicated genes is increased with dosage, but for others it appears that some form of dosage compensation occurs.
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Project description:Cyclin Dependent Kinases CDK8 and CDK19 (Mediator kinase) are regulatory components of the Mediator complex, a highly conserved complex that fine tunes transcriptional output. While Mediator kinase has been implicated in the transcriptional control of key pathways necessary for development and growth, its function in vivo has not been well described. Herein, we report the consequences of complete ablation of both Cdk8/19 on tissue homeostasis. We show that intestinal epithelial specific deletion of Mediator kinase leads to a distinct defect in secretory progenitor differentiation with broad loss of the intestinal secretory cell types. Using a phospho-proteogenomic approach, we show that the Cdk8/19 kinase module interacts with and phosphorylates components of the chromatin remodeling complex Swi/Snf in intestinal epithelial cells. Genomic localisation of Swi/Snf and Mediator shows Cdk8/19-dependent genomic binding at distinct super-enhancer loci within key lineage specification genes, including the master regulator of secretory differentiation ATOH1. Using CRISPRi/CRISPRa, we identify a distinct Mediator-Swi/Snf bound enhancer element that is necessary and sufficient for ATOH1 expression in a Mediator-kinase dependent manner. As such, these studies uncover a newly described transcriptional mechanism of ATOH1-dependent intestinal cell specification that is dependent on the coordinated interaction of the chromatin remodeling complex Swi/Snf and Mediator complex.
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Project description:Cytokines are highly pleiotropic ligands that critically contribute to a balanced immune response. We have an incomplete understanding of how cytokines elicit their functional pleiotropy, which has limited their therapeutic efficacy. Here, using Interleukin-6 (IL-6) as a model system, we have performed detailed phosphoproteomic and transcriptomic studies in human CD4 + T helper (Th)-1 cells to address the molecular bases defining cytokine functional pleiotropy. We have identified cyclin dependent kinase (CDK)8 as a new negative regulator of transcriptional activities of signal transducer and activator of transcription (STAT)3. We found that CDK8 is a major regulator of the IL-6 phosphoproteome and interacts with STAT3 in the nucleus upon IL-6 stimulation. Inhibition of CDK8 activity, using specific small molecules inhibitors, reduced the IL-6-induced phosphoproteome by 23% in Th-1 cells, including STAT3 S727 phosphorylation. STAT3 binding to target DNA sites in the genome was increased upon CDK8 inhibition, which resulted in a concomitant increase in STAT3 mediated transcriptional activity. Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions resulted in an enhancement of Th-17 differentiation. Our results support a model where CDK8 regulates STAT3 transcriptional processivity via modulation of its gene loci resident time, critically contributing to diversification of IL-6 responses.
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Project description:Cyclin C was cloned as a growth-promoting G1 cyclin1,2, and several studies postulated a role for cyclin C in driving cell proliferation3-8 . Moreover, cyclin C, together with its kinase partner, the cyclin-dependent kinase CDK8, is believed to represent an essential component of basal transcriptional machinery where it globally represses gene expression9-13. However, the function of cyclin C in vivo has never been addressed. Here we show that in the living organism cyclin C acts as a haploinsufficient tumor suppressor, through its function of controlling Notch1 oncogene levels. Cyclin C activates an “orphan” CDK19 kinase14, as well as CDK8 and CDK3. These cyclin C-CDK complexes phosphorylate Notch1 intracellular domain (ICN1), which allows binding of ICN1 to Fbw7 and triggers ICN1 polyubiquitination. Genetic ablation of cyclin C blocks ICN1 phosphorylation, disrupts Fbw7 binding, and decreases ICN1 ubiquitination in vivo, thereby strongly elevating ICN1 levels in several compartments of cyclin C knockout mice. Cyclin C was cloned as a growth-promoting G1 cyclin1,2, and several studies postulated a role for cyclin C in driving cell proliferation3-8 . Moreover, cyclin C, together with its kinase partner, the cyclin-dependent kinase CDK8, is believed to represent an essential component of basal transcriptional machinery where it globally represses gene expression9-13. However, the function of cyclin C in vivo has never been addressed. Here we show that in the living organism cyclin C acts as a haploinsufficient tumor suppressor, through its function of controlling Notch1 oncogene levels. Cyclin C activates an “orphan” CDK19 kinase14, as well as CDK8 and CDK3. These cyclin C-CDK complexes phosphorylate Notch1 intracellular domain (ICN1), which allows binding of ICN1 to Fbw7 and triggers ICN1 polyubiquitination. Genetic ablation of cyclin C blocks ICN1 phosphorylation, disrupts Fbw7 binding, and decreases ICN1 ubiquitination in vivo, thereby strongly elevating ICN1 levels in several compartments of cyclin C knockout mice.
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Project description:The transposon silencing piRNAs are produced from precursors that are encoded by heterochromatic clusters and processed in the perinuclear nuage. We show that the Drosophila nuclear DEAD box protein UAP56, previously implicated in mRNA splicing and nuclear export, co-localizes with the cluster-associated HP1 homologue Rhino. Prominent nuclear foci containing Rhi and UAP56 localize directly across the nuclear envelope from Vasa, a conserved DEAD box protein and core nuage component that is required for piRNA production, and piRNA precursors immunoprecipitate with both UAP56 and Vasa. A uap56 point mutation that prevents UAP56 protein co-localization with Rhino also disrupts nuage organization, transposon silencing, and expression of dual strand piRNA clusters. By contrast, this allele significantly increases ectopic piRNAs from protein coding genes. We therefore propose that UAP56 and Vasa organize a piRNA-processing compartment that spans the nuclear envelope, increasing the efficiency and specificity of piRNA biogenesis. 3 replicates of each sample (uap56, vasa), total RNA samples hybridized to tiling array.
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Project description:Cyclin Dependent Kinases CDK8 and CDK19 (Mediator kinase) are regulatory components of the Mediator complex, a highly conserved complex that fine tunes transcriptional output. While Mediator kinase has been implicated in the transcriptional control of key pathways necessary for development and growth, its function in vivo has not been well described. Herein, we report the consequences of complete ablation of both Cdk8/19 on tissue homeostasis. We show that intestinal epithelial specific deletion of Mediator kinase leads to a distinct defect in secretory progenitor differentiation with broad loss of the intestinal secretory cell types. Using a phospho-proteogenomic approach, we show that the Cdk8/19 kinase module interacts with and phosphorylates components of the chromatin remodeling complex Swi/Snf in intestinal epithelial cells. Genomic localisation of Swi/Snf and Mediator shows Cdk8/19-dependent genomic binding at distinct super-enhancer loci within key lineage specification genes, including the master regulator of secretory differentiation ATOH1. Using CRISPRi/CRISPRa, we identify a distinct Mediator- Swi/Snf bound enhancer element that is necessary and sufficient for ATOH1 expression in a Mediator-kinase dependent manner. As such, these studies uncover a newly described transcriptional mechanism of ATOH1-dependent intestinal cell specification that is dependent on the coordinated interaction of the chromatin remodeling complex Swi/Snf and Mediator complex.
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