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A Med15 - Hrp1 complex associates with fission yeast Mediator

ABSTRACT: 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

ORGANISM(S): Schizosaccharomyces pombe Saccharomyces cerevisiae

PROVIDER: GSE10079 | GEO | 2010/09/21

SECONDARY ACCESSION(S): PRJNA108643

REPOSITORIES: GEO

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Schizosaccharomyces pombe

Project description:A Med15 - Hrp1 complex associates with fission yeast Mediator

| PRJNA108643 | ENA

The polymorphic PolyQ tail protein of the Mediator Complex, Med15, regulates variable response to stress

Project description:The Mediator is composed of multiple of proteins in the head, body, tail and CDK subunits conserved from yeast to humans. However, not all the components are required for transcription. Components of the tail subunit are not essential but to varying degrees are required for changes in transcription to stress. While some stresses are familiar such as heat, desiccation, and starvation, others are exotic yet elicit a physical stress response. MCHM is a hydrotrope that induces growth arrest in yeast. By exploiting genetic variation, specifically in Med15, between yeast strains, we found that Med15 with the polyQ expansion conferred MCHM sensitivity. This sensitivity was not from a loss of function as the reciprocal hemizygous hybrids were all sensitive, suggesting that there is an incompatibility between Mediator complexes from genetic divergent yeast strains. Transcriptomics from yeast expressing the incompatible Med15 changed expression in diverse pathways. Expansion of polyQ tracts in Med15 resulted in multiple isoforms which were mostly from likely post-translational modifications. Stability of Med15 was dependent on Ydj1, a chaperone and the incompatible Med15 allele was expressed at lower levels and less stable than the compatible Med15 allele. Med15 is tethered to the rest of the Mediator complex via Med2 and 3. Deletion of either Med2 or Med3 changes the Med15 isoform patterns in the in a similar manner whereas deletion of Med5, a distal component of the tail did not change the pattern. The med2 and 3 mutants are similarly sensitive to MCHM while med5 mutants are not. Differences between the Med15 alleles extend to responses to different stresses. Whereas the incompatible allele of Med15 improved growth to chemicals that produce free radicals, and the compatible allele of Med15 improved growth to reducing agents, caffeine, and hydroxyurea. This may reflect the positive and negative role that Med15 has in transcription.

2020-04-06 | GSE129898 | GEO

Transcriptional coactivator MED15 is required for beta cell maturation

Project description:Mediator, a co-regulator required for RNA pol II activity, interacts with tissue-specific transcription factors to regulate development and maintain homeostasis. We sought to understand whether Mediator subunit MED15 acts as a node that controls tissue-specific gene expression, using pancreatic insulin-producing β-cell maturation as a model. We found Med15 to be expressed during mouse pancreatic organogenesis and β-cell maturation; moreover, islets from human T2D donors feature reduced MED15 expression. Loss of Med15 in mouse β-cells caused defects in maturation without affecting β-cell mass or insulin expression. ChIP-seq and co-immunoprecipitation analyses determined that Med15 binds β-cell transcription factors Nkx6-1 and NeuroD1 to regulate key β-cell maturation genes. Human embryonic stem cell derived β-like cells, genetically engineered to express high levels of MED15, had increased maturation markers and improved insulin secretion. We provide the first evidence of the importance of Mediator in β-cell maturation and demonstrate an additional layer of control that tunes transcription factor function.

2024-09-10 | GSE137145 | GEO

The polymorphic PolyQ tail protein of the Mediator Complex, Med15, regulates variable response to stress

Project description:The polymorphic PolyQ tail protein of the Mediator Complex, Med15, regulates variable response to stress

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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

Functional studies of the Yeast Mediator Tail Module Subunits

2013-06-07 | GSE47712 | GEO

A genome wide role for CHD remodeling factors and Nap1 in nucleosome disassembly

Project description:Chromatin remodeling factors and histone chaperones were previously shown to cooperatively affect nucleosome assembly and disassembly processes in vitro. Here we show that S. pombe CHD remodellers, Hrp1 and Hrp3 physically interact with the histone chaperone Nap1. Genome wide analysis of Hrp1, Hrp3 and Nap1 occupancy, combined with nucleosome density measurements in respective mutants revealed that the CHD factors and Nap1 co-localized in particular to promoter regions where they remove nucleosomes near the transcriptional start site. Hrp1 and Hrp3 also regulate nucleosome density in coding regions where they have redundant roles to stimulate transcription. Previously, DNA replication dependent and independent nucleosome disassembly processes have been described. We found that nucleosome density increased in the hrp1 mutant in the absence of DNA replication. Finally, regions where nucleosome density increased in hrp1, hrp3 and nap1 mutants also showed nucleosome density and histone modification changes in HDAC and HAT mutants. Thus, this study revealed an important in vivo role for CHD remodellers and Nap1 in nucleosome disassembly at promoters and coding regions, which are linked to changes in histone acetylation. Keywords: ChIP on CHIP and expression profiling

2007-05-19 | GSE6557 | GEO

Genome-wide studies of histone Me2 SWIRM and polyamine oxidase domain homologues of mammalian LSD1

Project description:In order to gain a more global view of the activity of histone demethylases we report here studies of the two fission yeast SWIRM and polyamine oxidase domain homologues of mammalian LSD1. Consistent with previous work (Nicolas et al., 2006) we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. We find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) to up- and down-regulate expression of different genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1+ gene. Using hyper-geometric probability comparisons we uncover links between lysine specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. Keywords: chip on chip Genome-wide studies of histone demethylation catalysed by the fission yeast SWIRM and polyamine oxidase domain homologues of mammalian LSD1

2010-05-27 | E-GEOD-6224 | biostudies-arrayexpress

ChIP-seq of Mediator subunits in Mediator mutants and wild type yeast

Project description:Mediator subunits Med17 (Srb4) and Med15 (Gal11) were subjected to ChIP in wild type yeast, med18 (srb5), med20 (srb2), and med2 med3 med15 (triple mutant) yeast, all in kin28-anchor away yeast after 1 hr rapamycin treatment to evict Kin28 from the nucleus. This results in inactivation of Kin28, which reduces Mediator turnover at promoters and allows stronger Mediator ChIP signal at promoters. <br></br>Both Med17 and Med15 were tagged with myc and precipitated using the monoclonal antibody against c-myc. Please note that a specific control for rapamycin treatment was not performed in this experiment, as that has been well documented in the literature for this experiment, using what is called the anchor away method. The untagged sample is a control for non-specific immunoprecipitation.

2017-12-01 | E-MTAB-5824 | biostudies-arrayexpress

Effect of loss of function of Gal11/Med15 and Med3 from the Mediator tail module in budding yeast

Project description:Gene expression was compared for wild type yeast (BY4741) and yeast lacking Gal11/Med15 and Med3, or from a gal11-myc med3â strain. The gal11-myc allele shows a partial loss of function when combined with med3â. Expression was analyzed for yeast grown in YPD as well as in CSM. We also examined gene expression of the wild type strain BY4742 grown in YPD and include that data here. Gene expression was compared for wild type yeast (BY4741 and BY4742) and yeast lacking Gal11/Med15 and Med3, or from a gal11-myc med3â strain.

2011-08-31 | E-GEOD-31774 | biostudies-arrayexpress

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