Functional interplay between Mediator and TFIIB in preinitiation complex assembly in relation to promoter architecture.

Mediator is a large coregulator complex conserved from yeast to humans and involved in many human diseases, including cancers. Together with general transcription factors, it stimulates preinitiation complex (PIC) formation and activates RNA polymerase II (Pol II) transcription. In this study, we analyzed how Mediator acts in PIC assembly using in vivo, in vitro, and in silico approaches. We revealed an essential function of the Mediator middle module exerted through its Med10 subunit, implicating a key interaction between Mediator and TFIIB. We showed that this Mediator-TFIIB link has a global role on PIC assembly genome-wide. Moreover, the amplitude of Mediator's effect on PIC formation is gene-dependent and is related to the promoter architecture in terms of TATA elements, nucleosome occupancy, and dynamics. This study thus provides mechanistic insights into the coordinated function of Mediator and TFIIB in PIC assembly in different chromatin contexts.

Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo.

Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway.

Mediator-dependent recruitment of TFIIH modules in preinitiation complex.

In vitro, without Mediator, the association of general transcription factors (GTF) and RNA polymerase II (Pol II) in preinitiation complexes (PIC) occurs in an orderly fashion. In this work, we explore the in vivo function of Mediator in GTF recruitment to PIC. A direct interaction between Med11 Mediator head subunit and Rad3 TFIIH subunit was identified. We explored the significance of this interaction and those of Med11 with head module subunits Med17 and Med22 and found that impairing these interactions could differentially affect the recruitment of TFIIH, TFIIE, and Pol II in the PIC. A med11 mutation that altered promoter occupancy by the TFIIK kinase module of TFIIH genome-wide also reduced Pol II CTD serine 5 phosphorylation. We conclude that the Mediator head module plays a critical role in TFIIH and TFIIE recruitment to the PIC. We identify steps in PIC formation that suggest a branched assembly pathway.

A high resolution protein interaction map of the yeast Mediator complex.

Mediator is a large, modular protein complex remotely conserved from yeast to man that conveys regulatory signals from DNA-binding transcription factors to RNA polymerase II. In Saccharomyces cerevisiae, Mediator is thought to be composed of 24 subunits organized in four sub-complexes, termed the head, middle, tail and Cdk8 (Srb8-11) modules. In this work, we have used screening and pair-wise two-hybrid approaches to investigate protein-protein contacts between budding yeast Mediator subunits. The derived interaction map includes the delineation of numerous interaction domains between Mediator subunits, frequently corresponding to segments that have been conserved in evolution, as well as novel connections between the Cdk8 (Srb8-11) and head modules, the head and middle modules, and the middle and tail modules. The two-hybrid analysis, together with co-immunoprecipitation studies and gel filtration experiments revealed that Med31 (Soh1) is associated with the yeast Mediator that therefore comprises 25 subunits. Finally, analysis of the protein interaction network within the Drosophila Mediator middle module indicated that the structural organization of the Mediator complex is conserved from yeast to metazoans. The resulting interaction map provides a framework for delineating Mediator structure-function and investigating how Mediator function is regulated.

Direct interaction of RNA polymerase II and mediator required for transcription in vivo.

Gene transcription is highly regulated. Altered transcription can lead to cancer or developmental diseases. Mediator, a multisubunit complex conserved among eukaryotes, is generally required for RNA polymerase II (Pol II) transcription. An interaction between the two complexes is known, but its molecular nature and physiological role are unclear. We identify a direct physical interaction between the Rpb3 Pol II subunit of Saccharomyces cerevisiae and the essential Mediator subunit, Med17. Furthermore, we demonstrate a functional element in the Mediator-Pol II interface that is important for genome-wide Pol II recruitment in vivo. Our findings suggest that a direct interaction between Mediator and Pol II is generally required for transcription of class II genes in eukaryotes.

Distinct roles for Mediator Cdk8 module subunits in Drosophila development.

Mediator (MED) is a conserved multisubunit complex bridging transcriptional activators and repressors to the general RNA polymerase II initiation machinery. In yeast, MED is organized in three core modules and a separable 'Cdk8 module' consisting of the cyclin-dependent kinase Cdk8, its partner CycC, Med12 and Med13. This regulatory module, specifically required for cellular adaptation to environmental cues, is thought to act through the Cdk8 kinase activity. Here we have investigated the functions of the four Cdk8 module subunits in the metazoan model Drosophila. Physical interactions detected among the four fly subunits provide support for a structurally conserved Cdk8 module. We analyzed the in vivo functions of this module using null mutants for Cdk8, CycC, Med12 and Med13. Each gene is required for the viability of the organism but not of the cell. Cdk8-CycC and Med12-Med13 act as pairs, which share some functions but also have distinct roles in developmental gene regulation. These data reveal functional attributes of the Cdk8 module, apart from its regulated kinase activity, that may contribute to the diversification of genetic programs.

Anc1 interacts with the catalytic subunits of the general transcription factors TFIID and TFIIF, the chromatin remodeling complexes RSC and INO80, and the histone acetyltransferase complex NuA3.

The Anc1 protein co-purifies with general transcription factors, chromatin remodeling complexes, and histone modification enzymes and is required for efficient transcription in yeast. We show here that Anc1 interacts with Tsm1, Tfg1, Sth1, Ino80, and Sas3 that are, respectively, the catalytic subunits of the general transcription factors TFIID and TFIIF, of the chromatin remodeling complexes RSC and INO80, and of the histone H3-acetyltransferase complex NuA3. We show that Anc1 is required for growth on galactose as the sole carbon source, and that it is recruited to the UAS of the GAL1 gene after induction.

Hmo1, an HMG-box protein, belongs to the yeast ribosomal DNA transcription system.

Hmo1 is one of seven HMG-box proteins of Saccharo myces cerevisiae. Null mutants have a limited effect on growth. Hmo1 overexpression suppresses rpa49-Delta mutants lacking Rpa49, a non-essential but conserved subunit of RNA polymerase I corresponding to the animal RNA polymerase I factor PAF53. This overexpression strongly increases de novo rRNA synthesis. rpa49-Delta hmo1-Delta double mutants are lethal, and this lethality is bypassed when RNA polymerase II synthesizes rRNA. Hmo1 co-localizes with Fob1, a known rDNA-binding protein, defining a narrow territory adjacent to the nucleoplasm that could delineate the rDNA nucleolar domain. These data identify Hmo1 as a genuine RNA polymerase I factor acting synergistically with Rpa49. As an HMG-box protein, Hmo1 is remotely related to animal UBF factors. hmo1-Delta and rpa49-Delta are lethal with top3-Delta DNA topoisomerase (type I) mutants and are suppressed in mutants lacking the Sgs1 DNA helicase. They are not affected by top1-Delta defective in Top1, the other eukaryotic type I topoisomerase. Conversely, rpa34-Delta mutants lacking Rpa34, a non-essential subunit associated with Rpa49, are lethal in top1-Delta but not in top3-Delta.