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Identification of Med26 target genes in Human HEK 293T cells

ABSTRACT: Med26 is a subunit of the Human Mediator complex. The Mediator complex is an evolutionarily conserved coregulatory complex that interacts with RNA polymerase II to regulate gene expression. In metazoa, Mediator is composed of some 30 distinct subunits. Mediator exists in multiple, functionally distinct forms that share a common core of subunits and can be distinguished by the presence or absence of a kinase module composed of Med12, Med13, Cdk8, and Cyclin C. In higher eukaryotes, a subset of Mediator complexes is associated with an additional subunit, Med26. This Med26-containing Mediator copurifies from cells with little or no kinase module, but near-stoichiometric Pol II. Evidence suggests that Med26-containing Mediator plays a key role in transcriptional activation however, the mechanism(s) by which Med26 contributes to this process are not known. To identify Med26 target genes, we used Affymetrix U133A plus 2.0 expression arrays to analyze mRNA expression in 293T cells from which Med26 had been depleted by transient transfection by each of three different siRNAs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE28715 | GEO | 2011/09/01

SECONDARY ACCESSION(S): PRJNA139013

REPOSITORIES: GEO

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Identification of Med26 target genes in Human HEK 293T cells

Project description:Med26 is a subunit of the Human Mediator complex. The Mediator complex is an evolutionarily conserved coregulatory complex that interacts with RNA polymerase II to regulate gene expression. In metazoa, Mediator is composed of some 30 distinct subunits. Mediator exists in multiple, functionally distinct forms that share a common core of subunits and can be distinguished by the presence or absence of a kinase module composed of Med12, Med13, Cdk8, and Cyclin C. In higher eukaryotes, a subset of Mediator complexes is associated with an additional subunit, Med26. This Med26-containing Mediator copurifies from cells with little or no kinase module, but near-stoichiometric Pol II. Evidence suggests that Med26-containing Mediator plays a key role in transcriptional activation however, the mechanism(s) by which Med26 contributes to this process are not known. To identify Med26 target genes, we used Affymetrix U133A plus 2.0 expression arrays to analyze mRNA expression in 293T cells from which Med26 had been depleted by transient transfection by each of three different siRNAs. Three different independent Med26 siRNA knockdowns were compared to a non-targeting control in triplicate, for a total of 12 samples.

2011-08-31 | E-GEOD-28715 | biostudies-arrayexpress

Requirement for the Mediator kinase module for activation of super-enhancer associated genes in colon cancer cells

Project description:RNA sequencing of HCT116 colon cancer cells following single and combined depletions of Mediator kinase module subunits CDK8, CDK19, MED12, MED13 and MED13L and the BET family protein BRD4. The results provide insight into the shared and specific functions of kinase module subunits and BRD4 in regulation of expression of transcriptional programs including genes associated with cancer acquired super-enhancers.

2018-04-02 | E-MTAB-5873 | biostudies-arrayexpress

The role of Mediator and Little Elongation Complex in transcription termination

Project description:Mediator is a coregulatory complex involved in regulating the transcription of Pol II-dependent genes. Metazoan Mediator subunit MED26 functions as a docking site for the ELL/EAF-containing Super Elongation Complex (SEC) and L ittle Elongation Complex (LEC), which regulate the expression of distinct genes. MED26 helps to recruit SEC to protein-coding genes including c-Myc and LEC to small nuclear RNA (snRNA) genes. However, why MED26 takes advantage of SEC or LEC to regulate different claases of genes is unclear. Here, we present evidence that MED26 recruits LEC to support optimal transcription termination at non-polyadenylated genes including snRNA and replication-dependent histone (RDH) genes. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and then LEC promotes 3’-end processing through the recruitment of Integrator or Heat labile factor to snRNA or RDH genes, respectively.

2020-01-21 | GSE121024 | GEO

A study of Mediator subunit enhancer-promoter binding

Project description:Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter, but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter.

2016-10-31 | GSE82082 | GEO

Cdk8, Cyclin C, Med12 or Med13 depletion effect on gene expression in Drosophila S2 cells

Project description:Expression profiling following depletion of Mediator Cdk8 module subunits Cdk8, Cyclin C (CycC), Med12 and Med13 72 hours after dsRNA treatment of Drosophila melanogaster S2 cells. Results provide insight into the role of individual Cdk8 module subunits in regulation of transcription. 22 samples. 2 Cdk8 dsRNA, 4 CycC dsRNA, 4 Med12 dsRNA, 4 Med13 dsRNA, 8 control samples including 4 Luciferase (Luc) dsRNA and 4 GFP dsRNA

2014-04-29 | E-GEOD-52343 | biostudies-arrayexpress

Cdk8, Cyclin C, Med12 or Med13 depletion effect on gene expression in Drosophila S2 cells

2014-04-29 | GSE52343 | GEO

Proteomic interactome of C. elegans Mediator complex subunit 28 (MDT-28) reveals predominant association with a restricted set of core Mediator subunits and an affinity to additional structural and enzymatic proteins

Project description:Transcription factors exert their regulatory potential on RNA Polymerase II machinery through a multiprotein complex called Mediator complex or Mediator. The Mediator complex integrates regulatory signals from cell regulatory cascades with the regulation by transcription factors. The Mediator complex consists of 25 subunits in Saccharomyces cerevisiae and 30 or more subunits in multicellular eukaryotes. Mediator subunit 28 (MED28), along with MED30, MED23, MED25 and MED26 belong to presumably evolutionarily new subunits that seem to be absent in unicellular eukaryotes and are likely to have evolved together with multicellularity and cell differentiation. Previously, we have shown that an originally uncharacterized predicted gene F28F8.5 is the true MED28 orthologue in Caenorhabditis elegans (MDT-28) and showed that it is involved in a spectrum of developmental processes. Here, we studied the proteomic interactome of MDT-28 edited as GFP::MDT-28 using Crispr/Cas9 technology or MDT-28::GFP expressed from extrachromosomal arrays in transgenic C. elegans exploiting the GFP-TRAP system and mass spectrometry. The results show that MDT-28 associates with the Head module subunits MDT-6, MDT-8, MDT-11, MDT-17, MDT-20, MDT-22 and MDT-30 and the Middle module subunit MDT-14. The analyses also identified additional proteins as preferential MDT-28 interactants including chromatin organizing proteins, structural proteins and enzymes. The results provide evidence for MDT-28 engagement in the Mediator Head Module and support the possibility of physical (direct or indirect) interaction of MDT-28 with additional proteins, reflecting a transcription-regulating potential of primarily structural and enzymatic proteins at the level of the Mediator complex.

2020-07-15 | PXD015552 | Pride

Cdk11-Cyclin L controls the assembly of the RNA polymerase II Mediator [expression]

Project description:The L-Mediator is a general co-activator of RNA Polymerase II transcription and is formed by the reversible association of the S-Mediator and the kinase module harbouring Cdk8. We describe the Cdk11-Lcp1 complex and show that its inactivation alter the global expression profiles in a very similar way than mutations of the kinase module. Cdk11 is broadly distributed onto chromatin and phosphorylate the Med27 and Med4 Mediator subunits on conserved residues. The inactivation of either Cdk11 or the mutation of its target residues on the Mediator leads to a strongly decreased association of the kinase module and the S-Mediator. The results show that Cdk11-Lcp1 regulates that assembly of the L-Mediator complex. Total RNA was isolated from two biological replicates for all conditions, and each biological replicate was hybridized in duplicate on Agilent arrays (dye-swap).

2014-07-11 | E-GEOD-37960 | biostudies-arrayexpress

Cdk11-Cyclin L controls the assembly of the RNA polymerase II Mediator [ChIP-chip]

Project description:The L-Mediator is a general co-activator of RNA Polymerase II transcription and is formed by the reversible association of the S-Mediator and the kinase module harbouring Cdk8. We describe the Cdk11-Lcp1 complex and show that its inactivation alter the global expression profiles in a very similar way than mutations of the kinase module. Cdk11 is broadly distributed onto chromatin and phosphorylate the Med27 and Med4 Mediator subunits on conserved residues. The inactivation of either Cdk11 or the mutation of its target residues on the Mediator leads to a strongly decreased association of the kinase module and the S-Mediator. The results show that Cdk11-Lcp1 regulates that assembly of the L-Mediator complex. Two independent immunoprecipitations were done for HA-tagged Cdk11 and each biological replicate was hybridized in duplicate on Agilent arrays using input DNA as the reference channel.

2014-07-11 | E-GEOD-37961 | biostudies-arrayexpress

Mediator12 and 13 act as conditional anti-silencing factors in Arabidopsis [ChIP-seq]

Project description:Mediator is a multi-protein complex which facilitates the initial steps of gene transcription. Mediator 12 (MED12) and MED13 are components of a module that reversibly associates with the core Mediator complex, and both positively and negatively regulates gene expression. Here, in an Arabidopsis mutant screen for factors that can bypass repressive epigenetic marks, we identified MED12 and MED13 as anti-silencing factors. Both are preferentially required for the expression of genes in the absence of H3K4me3, an activating chromatin mark. Thus, MED12 and MED13 respond to specific epigenetic states and can act as conditional positive gene regulators in Arabidopsis.

2020-12-31 | GSE123562 | GEO

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