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Mediator subunit Med15 dictates the conserved "fuzzy" binding mechanism of yeast transcription activators Gal4 and Gcn4.

ABSTRACT: The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a "fuzzy" protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two intrinsically disordered ADs of different sequence, interact nearly identically with Med15. The finding that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts strongly with Gal80 via a distinct structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein-protein interaction.

SUBMITTER: Tuttle LM

PROVIDER: S-EPMC8044209 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The acidic transcription activator Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex.

Project description:The structural basis for binding of the acidic transcription activator Gcn4 and one activator-binding domain of the Mediator subunit Gal11/Med15 was examined by NMR. Gal11 activator-binding domain 1 has a four-helix fold with a small shallow hydrophobic cleft at its center. In the bound complex, eight residues of Gcn4 adopt a helical conformation, allowing three Gcn4 aromatic/aliphatic residues to insert into the Gal11 cleft. The protein-protein interface is dynamic and surprisingly simple, involving only hydrophobic interactions. This allows Gcn4 to bind Gal11 in multiple conformations and orientations, an example of a "fuzzy" complex, where the Gcn4-Gal11 interface cannot be described by a single conformation. Gcn4 uses a similar mechanism to bind two other unrelated activator-binding domains. Functional studies in yeast show the importance of residues at the protein interface, define the minimal requirements for a functional activator, and suggest a mechanism by which activators bind to multiple unrelated targets.

| S-EPMC3246216 | biostudies-literature

The evolutionarily conserved mediator subunit MDT-15/MED15 links protective innate immune responses and xenobiotic detoxification.

Project description:Metazoans protect themselves from environmental toxins and virulent pathogens through detoxification and immune responses. We previously identified a small molecule xenobiotic toxin that extends survival of Caenorhabditis elegans infected with human bacterial pathogens by activating the conserved p38 MAP kinase PMK-1 host defense pathway. Here we investigate the cellular mechanisms that couple activation of a detoxification response to innate immunity. From an RNAi screen of 1,420 genes expressed in the C. elegans intestine, we identified the conserved Mediator subunit MDT-15/MED15 and 28 other gene inactivations that abrogate the induction of PMK-1-dependent immune effectors by this small molecule. We demonstrate that MDT-15/MED15 is required for the xenobiotic-induced expression of p38 MAP kinase PMK-1-dependent immune genes and protection from Pseudomonas aeruginosa infection. We also show that MDT-15 controls the induction of detoxification genes and functions to protect the host from bacteria-derived phenazine toxins. These data define a central role for MDT-15/MED15 in the coordination of xenobiotic detoxification and innate immune responses.

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Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex.

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Functional studies of the yeast med5, med15 and med16 mediator tail subunits.

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Formation of nuclear condensates by the Mediator complex subunit Med15 in mammalian cells.

Project description:BackgroundThe Mediator complex is an evolutionarily conserved multi-subunit protein complex that plays major roles in transcriptional activation and is essential for cell growth, proliferation, and differentiation. Recent studies revealed that some Mediator subunits formed nuclear condensates that may facilitate enhancer-promoter interactions and gene activation. The assembly, regulation, and functions of these nuclear condensates remain to be further understood.ResultsWe found that Med15, a subunit in the tail module of the Mediator complex, formed nuclear condensates through a novel mechanism. Nuclear foci of Med15 were detected by both immunostaining of endogenous proteins and live cell imaging. Like Med1 foci and many other biomolecular condensates, Med15 foci were sensitive to 1, 6-Hexanediol and showed rapid recovery during fluorescence recovery after photobleaching. Interestingly, overexpressing DYRK3, a dual-specificity kinase that controls the phase transition of membraneless organelles, appeared to disrupt Med1 foci and Med15 foci. We identified two regions that are required to form Med15 nuclear condensates: the glutamine-rich intrinsically disordered region (IDR) and a short downstream hydrophobic motif. The optodroplet assay revealed that both the IDR and the C-terminal region of Med15 contributed to intracellular phase separation.ConclusionsWe identified that the Mediator complex subunit Med15 formed nuclear condensates and characterized their features in living cells. Our work suggests that Med15 plays a role in the assembly of transcription coactivator condensates in the nucleus and identifies Med15 regions that contribute to phase separation.

| S-EPMC8597291 | biostudies-literature

Mediator Subunit Med15 Regulates Cell Morphology and Mating in <i>Candida lusitaniae</i>.

Project description:Candida lusitaniae is an emerging opportunistic pathogenic yeast capable of shifting from yeast to pseudohyphae form, and it is one of the few Candida species with the ability to reproduce sexually. In this study, we showed that a dpp3Δ mutant, inactivated for a putative pyrophosphatase, is impaired in cell separation, pseudohyphal growth and mating. The defective phenotypes were not restored after the reconstruction of a wild-type DPP3 locus, reinforcing the hypothesis of the presence of an additional mutation that we suspected in our previous study. Genetic crosses and genome sequencing identified an additional mutation in MED15, encoding a subunit of the mediator complex that functions as a general transcriptional co-activator in Eukaryotes. We confirmed that inactivation of MED15 was responsible for the defective phenotypes by rescuing the dpp3Δ mutant with a wild-type copy of MED15 and constructing a med15Δ knockout mutant that mimics the phenotypes of dpp3Δ in vitro. Proteomic analyses revealed the biological processes under the control of Med15 and involved in hyphal growth, cell separation and mating. This is the first description of the functions of MED15 in the regulation of hyphal growth, cell separation and mating, and the pathways involved in C. lusitaniae.

| S-EPMC10053558 | biostudies-literature

Activator Gcn4 employs multiple segments of Med15/Gal11, including the KIX domain, to recruit mediator to target genes in vivo.

Project description:Mediator is a multisubunit coactivator required for initiation by RNA polymerase II. The Mediator tail subdomain, containing Med15/Gal11, is a target of the activator Gcn4 in vivo, critical for recruitment of native Mediator or the Mediator tail subdomain present in sin4Delta cells. Although several Gal11 segments were previously shown to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We show that interaction of Gcn4 with the Mediator tail in vitro and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo involve additive contributions from three different segments in the N terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we establish that B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrate that Gcn4 binds to the Gal11 KIX domain directly and, by NMR chemical shift analysis combined with mutational studies, we identify the likely binding site for Gcn4 on the KIX surface. Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator in vivo.

| S-EPMC2807301 | biostudies-literature

A Trio of Binding Partners for Gcn4♦ : Activator Gcn4 Employs Multiple Segments of Med15/Gal11, Including the KIX Domain, to Recruit Mediator to Target Genes in Vivo

Project description: Not available

| S-EPMC2807343 | biostudies-literature

Genetic and physical interactions between Tel2 and the Med15 Mediator subunit in Saccharomyces cerevisiae.

Project description:BackgroundIn budding yeast, the highly conserved Tel2 protein is part of several complexes and its main function is now believed to be in the biogenesis of phosphatidyl inositol 3-kinase related kinases.Principal findingsTo uncover potentially novel functions of Tel2, we set out to isolate temperature-sensitive (ts) mutant alleles of TEL2 in order to perform genetic screenings. MED15/GAL11, a subunit of Mediator, a general regulator of transcription, was isolated as a suppressor of these mutants. The isolated tel2 mutants exhibited a short telomere phenotype that was partially rescued by MED15/GAL11 overexpression. The tel2-15 mutant was markedly deficient in the transcription of EST2, coding for the catalytic subunit of telomerase, potentially explaining the short telomere phenotype of this mutant. In parallel, a two-hybrid screen identified an association between Tel2 and Rvb2, a highly conserved member of the AAA+ family of ATPases further found by in vivo co-immunoprecipitation to be tight and constitutive. Transiently overproduced Tel2 and Med15/Gal11 associated together, suggesting a potential role for Tel2 in transcription. Other Mediator subunits, as well as SUA7/TFIIB, also rescued the tel2-ts mutants.SignificanceAltogether, the present data suggest the existence of a novel role for Tel2, namely in transcription, possibly in cooperation with Rvb2 and involving the existence of physical interactions with the Med15/Gal11 Mediator subunit.

| S-EPMC3265489 | biostudies-literature

A Med15 - Hrp1 complex associates with fission yeast Mediator

Project description:The multiprotein Mediator complex is an important regulator of RNA polymerase II-dependent genes in eukaryotic cell. In contrast to the situation in many other eukaryotes, the conserved Med15 protein is not a stable component of Mediator isolated from fission yeast. We now demonstrate that Med15 exists in a protein complex together with Hrp1, an ATP-dependent chromatin remodeling protein. The Med15/Hrp1 subcomplex is not a component of the core Mediator complex, but can interact with the repressive L-Mediator conformation. Deletion of MED15 and HRP1 cause similar effects on global steady-state levels of mRNA, but only MED15 is required for galactose-dependent activation of the inv1 gene. Hrp1 has been found in complex with other proteins and genome-wide analysis demonstrates that Med15 only associates with a distinct subset of Hrp1-bound gene promoters. Global analysis reveals that Hrp1-binding normally is associated with increased histone H3 density, but at promoters also bound by Med15, histone H3 density is instead increased. Our findings reveal that Med15 functions as a separate entity in fission yeast and indicate that the function and organization of the Mediator complex may differ significantly between eukaryotes. Keywords: ChIP-chip

2010-09-21 | GSE10079 | GEO

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