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Structure of mammalian Mediator complex reveals Tail module architecture and interaction with a conserved core.

ABSTRACT: The Mediator complex plays an essential and multi-faceted role in regulation of RNA polymerase II transcription in all eukaryotes. Structural analysis of yeast Mediator has provided an understanding of the conserved core of the complex and its interaction with RNA polymerase II but failed to reveal the structure of the Tail module that contains most subunits targeted by activators and repressors. Here we present a molecular model of mammalian (Mus musculus) Mediator, derived from a 4.0 Å resolution cryo-EM map of the complex. The mammalian Mediator structure reveals that the previously unresolved Tail module, which includes a number of metazoan specific subunits, interacts extensively with core Mediator and has the potential to influence its conformation and interactions.

SUBMITTER: Zhao H

PROVIDER: S-EPMC7921410 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Structure of mammalian Mediator complex reveals Tail module architecture and interaction with a conserved core.

Zhao Haiyan H Young Natalie N Kalchschmidt Jens J Lieberman Jenna J El Khattabi Laila L Casellas Rafael R Asturias Francisco J FJ

Nature communications 20210301 1

The Mediator complex plays an essential and multi-faceted role in regulation of RNA polymerase II transcription in all eukaryotes. Structural analysis of yeast Mediator has provided an understanding of the conserved core of the complex and its interaction with RNA polymerase II but failed to reveal the structure of the Tail module that contains most subunits targeted by activators and repressors. Here we present a molecular model of mammalian (Mus musculus) Mediator, derived from a 4.0 Å resolut ...[more]

PMID: 33649303

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A conserved Mediator-CDK8 kinase module association regulates Mediator-RNA polymerase II interaction.

Project description:The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) C-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a pre-eminent complex in eukaryotic transcription regulation. We used macromolecular EM and biochemistry to investigate the subunit organization, structure and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator's middle module interferes with CTD-dependent RNAPII binding to a previously unknown middle-module CTD-binding site and with the holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the mechanism of transcription regulation by Mediator.

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Mammalian mediator structure and conformation regulation by the Tail module

Project description:The Mediator complex plays an essential and multi-faceted role in regulation of RNA polymerase II transcription in all eukaryotes. Structural analysis of yeast Mediator provided an understanding of the conserved core of the complex and its interaction with RNA polymerase II, but failed to reveal how Mediator might enable a response to interaction with transcription factors (TFs). Here we present an atomic model of mammalian (Mus musculus) Mediator, derived from a 4.0 Å resolution cryo-EM map of the complex. The mammalian Mediator structure reveals how the previously unresolved Tail module, which includes a number of metazoan specific subunits, modulates Mediator conformation and interactions. The structure also suggests how a bi-partite organization of TF-interacting subunits can be exploited to enable a Mediator mechanism that combines conformational control of molecular interactions and enhancement of transcription through phase separation and enhancer-specific recruitment

2020-03-30 | GSE145902 | GEO

The tail-module of yeast Mediator complex is required for telomere heterochromatin maintenance.

Project description:Eukaryotic chromosome ends have a DNA-protein complex structure termed telomere. Integrity of telomeres is essential for cell proliferation. Genome-wide screenings for telomere length maintenance genes identified several components of the transcriptional regulator, the Mediator complex. Our work provides evidence that Mediator is involved in telomere length regulation and telomere heterochromatin maintenance. Tail module of Mediator is required for telomere silencing by promoting or stabilizing Sir protein binding and spreading on telomeres. Mediator binds on telomere and may be a component of telomeric chromatin. Our study reveals a specific role of Mediator complex at the heterochromatic telomere and this function is specific to telomeres as it has no effect on the HMR locus.

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Architecture of the Mediator head module.

Project description:Mediator is a key regulator of eukaryotic transcription, connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II). In the yeast Saccharomyces cerevisiae, Mediator comprises 25 subunits with a total mass of more than one megadalton (refs 5, 6) and is organized into three modules, called head, middle/arm and tail. Our understanding of Mediator assembly and its role in regulating transcription has been impeded so far by limited structural information. Here we report the crystal structure of the essential Mediator head module (seven subunits, with a mass of 223 kilodaltons) at a resolution of 4.3 ångströms. Our structure reveals three distinct domains, with the integrity of the complex centred on a bundle of ten helices from five different head subunits. An intricate pattern of interactions within this helical bundle ensures the stable assembly of the head subunits and provides the binding sites for general transcription factors and Pol II. Our structural and functional data suggest that the head module juxtaposes transcription factor IIH and the carboxy-terminal domain of the largest subunit of Pol II, thereby facilitating phosphorylation of the carboxy-terminal domain of Pol II. Our results reveal architectural principles underlying the role of Mediator in the regulation of gene expression.

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Mediator complex proximal Tail subunit MED30 is critical for Mediator core stability and cardiomyocyte transcriptional network.

Project description:Dysregulation of cardiac transcription programs has been identified in patients and families with heart failure, as well as those with morphological and functional forms of congenital heart defects. Mediator is a multi-subunit complex that plays a central role in transcription initiation by integrating regulatory signals from gene-specific transcriptional activators to RNA polymerase II (Pol II). Recently, Mediator subunit 30 (MED30), a metazoan specific Mediator subunit, has been associated with Langer-Giedion syndrome (LGS) Type II and Cornelia de Lange syndrome-4 (CDLS4), characterized by several abnormalities including congenital heart defects. A point mutation in MED30 has been identified in mouse and is associated with mitochondrial cardiomyopathy. Very recent structural analyses of Mediator revealed that MED30 localizes to the proximal Tail, anchoring Head and Tail modules, thus potentially influencing stability of the Mediator core. However, in vivo cellular and physiological roles of MED30 in maintaining Mediator core integrity remain to be tested. Here, we report that deletion of MED30 in embryonic or adult cardiomyocytes caused rapid development of cardiac defects and lethality. Importantly, cardiomyocyte specific ablation of MED30 destabilized Mediator core subunits, while the kinase module was preserved, demonstrating an essential role of MED30 in stability of the overall Mediator complex. RNAseq analyses of constitutive cardiomyocyte specific Med30 knockout (cKO) embryonic hearts and inducible cardiomyocyte specific Med30 knockout (icKO) adult cardiomyocytes further revealed critical transcription networks in cardiomyocytes controlled by Mediator. Taken together, our results demonstrated that MED30 is essential for Mediator stability and transcriptional networks in both developing and adult cardiomyocytes. Our results affirm the key role of proximal Tail modular subunits in maintaining core Mediator stability in vivo.

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Architecture of the RNA polymerase II-Mediator core transcription initiation complex

Project description:Comparative Dynamic Transcriptome Analysis (cDTA) enables global analysis of newly synthesized RNA as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111) and reveals defects in transcription with much higher sensitivity than conventional steady-state methods. cDTA was carried out as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111), using the S. cerevisiae heterozygous Med17/med17delta strain (Euroscarf) transfected with plasmids pRS315-SRB4 or pRS315-srb4-ts as described in Larivi̬re et al. Nature. 2012 (DOI:10.1038/nature11670), and Y40343-wildtype (Euroscarf) or Med18-FRB-KanMX6 (Euroscarf) strains. Heatshock of SRB4 and srb4-ts strains was applied for 18 or 60 minutes at 37C prior to RNA labeling as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111). To deplete the Med18 subunit from the nucleus, anchor-away experiments were performed by rapamycin treatment (1 ug/ml in 200 mL YPD) for 18 or 60 minutes at 30C prior to RNA labeling as described in Sun et al. Mol. Cell. 2013 (DOI:10.1016/j.molcel.2013.09.010). Data analysis was as described in Sun et al. Genome Res. 2012 (DOI:10.1101/gr.130161.111).

2015-02-04 | E-MTAB-2942 | biostudies-arrayexpress

Architecture of the budding yeast kinetochore reveals a conserved molecular core.

Project description:How kinetochore proteins are organized to connect chromosomes to spindle microtubules, and whether any structural and organizational themes are common to kinetochores from distantly related organisms, are key unanswered questions. Here, we used affinity chromatography and mass spectrometry to generate a map of kinetochore protein interactions. The budding yeast CENP-C homologue Mif2p specifically copurified with histones H2A, H2B, and H4, and with the histone H3-like CENP-A homologue Cse4p, strongly suggesting that Cse4p replaces histone H3 in a specialized centromeric nucleosome. A novel four-protein Mtw1 complex, the Nnf1p subunit of which has homology to the vertebrate kinetochore protein CENP-H, also copurified with Mif2p and a variety of central kinetochore proteins. We show that Mif2 is a critical in vivo target of the Aurora kinase Ipl1p. Chromatin immunoprecipitation studies demonstrated the biological relevance of these associations. We propose that a molecular core consisting of CENP-A, -C, -H, and Ndc80/HEC has been conserved from yeast to humans to link centromeres to spindle microtubules.

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The Cdk8 kinase module regulates interaction of the mediator complex with RNA polymerase II.

Project description:The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex mediator, which regulates gene transcription by RNA polymerase II. The CKM has both negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. In order to reconstitute the role of the CKM in transcription initiation, we prepared recombinant CKM from the yeast Saccharomyces cerevisiae. We showed that CKM bound to the core mediator (cMed) complex, sterically inhibiting cMed from binding to the polymerase II preinitiation complex (PIC) in vitro. We further showed that the Cdk8 kinase activity of the CKM weakened CKM-cMed interaction, thereby facilitating dissociation of the CKM and enabling mediator to bind the PIC in order to stimulate transcription initiation. Finally, we report that the kinase activity of Cdk8 is required for gene activation during the stressful condition of heat shock in vivo but not under steady-state growth conditions. Based on these results, we propose a model in which the CKM negatively regulates mediator function at upstream-activating sequences by preventing mediator binding to the PIC at the gene promoter. However, during gene activation in response to stress, the Cdk8 kinase activity of the CKM may release mediator and allow its binding to the PIC, thereby accounting for the positive function of CKM. This may impart improved adaptability to stress by allowing a rapid transcriptional response to environmental changes, and we speculate that a similar mechanism in metazoans may allow the precise timing of developmental transcription programs.

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Functional studies of the Yeast Mediator Tail Module Subunits

Project description:The yeast Mediator complex can be divided into three modules, designated Head, Middle and Tail. Tail comprises the Med2, Med3, Med5, Med15 and Med16 protein subunits, which are all encoded by genes that are individually non-essential for viability. In cells lacking Med16, Tail is displaced from Head and Middle. However, inactivation of MED5/MED15 and MED15/MED16 are synthetically lethal, indicating that Tail performs essential functions as a separate complex even when it is not bound to Middle and Head. We have used the N-Degron method to create temperature sensitive (ts) mutants in the Mediator tail subunits Med5, Med15 and Med16 to study the immediate effects on global gene expression when each subunit is individually inactivated, and when MED5/15 or MED15/16 are inactivated together. All Degron constructs were expressed from their normal chromosomal location under the control of their respective endogenous promoters. We isolated RNA from each strain as early as 45 minutes after changing from the permissive to the restrictive growth conditions to minimize possible secondary effects on gene expression that are not directly caused by the Degron construct(s).

2013-06-07 | E-GEOD-47712 | biostudies-arrayexpress

Mammalian mediator structure and conformation regulation by the Tail module

Project description:Mammalian mediator structure and conformation regulation by the Tail module

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