Mediator is a big multi-subunit complex highly conserved through all eukaryotes. The Mediator complex is made of 21 subunits in yeast and 26 in human. Indeed, the metazoan Mediator contains five additional subunits named MED23/25/26/28/30. The Mediator complex is an important component of the PIC and acts as a structural bridge between specific TFs and the RNAPII. It is organized in four distinct modules named the tail, the middle, the head and the kinase module. The
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Tail is composed of the subunits Med2, Med3, Med5/Nut1, Med15/Gal11 and Med16/Sin4, none of which is required for yeast cell viability, and historically it is considered the part of Mediator that interacts with the TFs at the UAS. The Middle comprises the subunits Med1, Med4, Med7, Med10, Med14, Med19/Rox3, Med21/Srb7 and Med31 and connects the Tail with the Head that is the part of Mediator more proximal to RNAPII. The Head is composed of the subunits Med6, Med8, Med11, Med17/Srb4, Med18/Srb5, Med20/Srb2 and Med22/Srb6. The kinase module comprises the kinase Cdk8, CyC, Med12 and Med13. Contrary to the other three modules, the kinase module is a dissociable module that transiently interacts with Mediator and regulates its association with RNAPII (see below) (reviewed in (Allen and Taatjes, 2015; Soutourina, 2018)). Importantly, all subunits of Mediator essential for cell viability reside in the middle and in the head modules underscoring the importance of these two modules (Figure 16).
Figure 16. Schematic representation of yeast and mammalian Mediator complexes. The asterisks label the subunits essential for yeast cell viability (Soutourina, 2018).
50 Mediator structure
Though in the 2000s the first crystal structures of some essential Mediator subunits became available (Baumli et al., 2005; Hoeppner et al., 2005; Koschubs et al., 2009; Lariviere et al., 2008), structural studies on Mediator modules or on the whole complex resulted to be challenging due to the high flexibility of the complex and the large number of disordered regions. The biggest contribution to define the structure of Mediator came from the work of Cramer and Kornberg’s groups that in 2012 independently published the cryo-EM structure of Mediator head module (Lariviere et al., 2012;
Robinson et al., 2012) and later of a minimal form of Mediator consisting of the middle and the head modules (Nozawa et al., 2017). These first structural studies revealed that Mediator has a triangle shape and is characterized by a high conformational flexibility. Mediator head has a crocodile-like shape characterized by the neck (or spine), the fixed jaw (or arm) and the moveable jaw. Mediator middle has an extended shape and is organized in four structural domains, the hook, the knob, the beam and the plank domains. The beam is entirely occupied by Med14, an essential subunit of the middle module, while the plank domains are made of Med4 and Med9. Med14 represents the structural center of Mediator, interacts with all the modules and is critical for preserving the architecture of the whole complex. Indeed, deletion of Med14 C-terminus results in disassembly of the tail module from the rest of Mediator (Cevher et al., 2014). Furthermore, Med14 extensively interacts with the Head essential subunits Med17 and Med6. Importantly, the interface between the Head and the Middle is very flexible and many Mediator mutants defective in transcription previously identified map to this region, hinting to the importance of Middle and Head interaction for transcription (reviewed in (Harper and Taatjes, 2017)).
A tremendous advance in understanding the structure and the function of Mediator in PIC assembly came from two outstanding structural studies published independently few years ago again by Cramer and Kornberg’s groups (Plaschka et al., 2015; Robinson et al., 2016). These two works reported the first cryo-EM structure of the Mediator complex associated to RNAPII. The two works
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confirmed the high conformational flexibility of the Mediator complex and revealed that it undergoes massive structural shifts upon binding to RNAPII and interaction with TFs (reviewed in (Plaschka et al., 2016b)). In the work from Plaschka et al. a minimal form of Mediator named core Mediator (cMed) and containing 15 subunits from the head and the middle modules was co-purified with RNAPII. Structure of the cITC (core initiation complex, lacking TFIIE and TFIIH) together with c-Med revealed the existence of three main interfaces, named A, B and C, between Mediator and the cITC.
The interface A, made by the interaction between Mediator moveable jaw, TFIIB and the RNAPII dock domain occupied by Rpb3 and Rpb11, suggests a central role of Mediator head in TFIIB stabilization and in the first phases of PIC assembly. The essential proteins Med17 and Med8 interact with the RNAPII subunit Rpb4 at the interface B. The importance of this interface is underscored by the observation that the mutation of the Med17-temperature-sensitive allele that results in a drastic drop in transcription (Plaschka et al., 2015) maps at this interface. The third interface between Mediator and RNAPII involves Mediator middle module. Indeed, the plank domains constitute by Med4 and Med9 interact with RNAPII foot (Figure 17). Importantly, this interaction is transient and is lost when TFIIS binds the PIC and elongation takes place. The cryo-EM structure of the whole Mediator complex associated with RNAPII solved by Kornberg’s lab confirmed the strong interaction between Mediator Head and RNAPII and highlighted the important role of RNAPII CTD. Indeed, removal of RNAPII CTD decreases Mediator association to RNAPII, suggesting that Mediator interacts with RNAPII CTD (Robinson et al., 2016). Importantly, the dissociable kinase module of Mediator could modulate Mediator head interaction with RNAPII CTD. Indeed, Robinson et al. showed that association of the Cdk8 module to core-Mediator, and more precisely the interaction between Cdk8 and Med13 in the hook domain, leads to a conformational change that occludes the surface interaction between Mediator head and the CTD. This structural observation is consistent with the observation that Cdk8 and RNAPII binding to Mediator are mutually exclusive (Elmlund et al., 2006;
Tsai et al., 2013).
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Finally, though the whole Mediator structure has been solved, a high-resolution structure of Mediator tail is still missing. However, contrary to the standard view of Mediator tail localized far away from RNAPII, the available low resolution studies seem to localize the Tail in proximity to the RNAPII subunits Rpb3 and Rpb11 (Robinson et al., 2016; Tsai et al., 2014).
Figure 17. (A) EM reconstruction of cITC and core Meditor (Plaschka et al., 2015). (B) Current model of Mediator interaction with the PIC as presented in (Plaschka et al., 2016b).
Mediator role in transcription regulation and beyond
The importance of the Mediator complex in transcription is underscored by the embryonic lethality of some Mediator null-mutants in metazoans (reviewed in (Yin and Wang, 2014)). In yeast, the transcription profiles of the temperature-sensitive mutants for Med14 and Med17 are highly comparable to Rbp1-temparature-sensitive profile and are characterized by a massive drop of transcription genome-wide (Holstege et al., 1998; Plaschka et al., 2015). Recent structural and molecular studies highlighted that Mediator plays a part in transcription initiation through several
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distinct mechanisms (reviewed in (Jeronimo and Robert, 2017)). First, the Mediator complex represents a physical scaffold for the assembly of the PIC. Indeed, Mediator stabilizes the PIC through multiple interactions with RNAPII (Plaschka et al., 2015; Robinson et al., 2016), TFIIB (Plaschka et al., 2015), TFIID (Koleske et al., 1992; Lariviere et al., 2006) and TBP (Lariviere et al., 2006). Consistently, with a main role of Mediator in PIC assembly, several Med17 and Med10 temperature-sensitive strains are characterized by reduced binding of TFIIB and TBP on some target genes (Eyboulet et al., 2015; Eychenne et al., 2016). Furthermore, the interaction between Mediator head and RNAPII CTD has a fundamental role in RNAPII promoter escape. A nice work from Robert’s lab showed indeed that phosphorylation of Ser5 on RNAPII CTD by Kin28 disrupts the interaction between Mediator and RNAPII favoring RNAPII release from the promoter and transcription elongation (Jeronimo and Robert, 2014).
Aside of its primary role in PIC assembly and RNAPII promoter escape, in metazoans Mediator regulates several steps of the transcription cycle and it is the end point of different signaling pathways cascades. For example, the metazoan-specific subunit Med26 is involved in RNAPII pausing, while different subunits of metazoan Mediator play a role in transcription elongation and re-initiation and enhancer-promoter DNA looping (reviewed in (Allen and Taatjes, 2015)).
Furthermore, some recent studies showed that Mediator might play a role in nuclear processes other than transcription, such as DNA repair and maintenance of the chromatin architecture (reviewed in (Allen and Taatjes, 2015)). Indeed, it has been shown that yeast Mediator binds to boundaries (Chereji et al., 2017a; Hsieh et al., 2015) and interacts with the TREX-2 complex (Schneider et al., 2015) and with chromatin remodeling complexes (Ansari et al., 2014). Consistently with a structural function of Mediator in chromatin architecture, Mediator might play a role also in establishment of heterochromatin in S.pombe (Oya et al., 2013).
Finally, Mediator contains two enzymatic activities, the acetyltransferase Med15 in the middle module and the kinase Cdk8. While the acetyltransferase activity of Med15 has been observed just in
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vitro (Lorch et al., 2000), it is known that Cdk8 phosphorylates some TFs in vivo (Poss et al., 2016).
Furthermore, it has been shown that in vitro Cdk8 phosphorylates both Ser2 and Ser5 on RNAPII CTD (Liao et al., 1995). However, deletion of Cdk8/Srb10 does not affect CTD phosphorylation levels in yeast cells (Rodriguez et al., 2000). Thus, whether Cdk8 contributes to phosphorylation of CTD in vivo is still controversial (reviewed in (Galbraith et al., 2010)).
Role of Mediator tail and Cdk8 modules in transcription regulation
While the middle and the head of Mediator are crucial for transcription, the importance of the tail and the kinase modules in transcription is more elusive. As already mentioned, none of the subunits belonging to these modules are essential for viability of yeast cells.
The role of the kinase module in transcription has been matter of debate for long time. As mentioned in the previous paragraph, it is not clear whether Cdk8 participates in phosphorylation of RNAPII CTD in vivo. Furthermore, early studies on the kinase module suggested a repressive role of Cdk8 in transcription (Holstege et al., 1998). Two recent studies proposed a model according to which Mediator undergoes conformational changes upon PIC assembly that lead to ejection of the Cdk8 module (Jeronimo et al., 2016; Petrenko et al., 2016). Furthermore, structural studies suggest that release of the Cdk8 module favors Mediator head interaction with RNAPII and thus transcription (Robinson et al., 2016). According to this model Mediator kinase module has a regulatory role rather than a direct function in transcription.
The Tail has been long considered to be the module with a key role in Mediator recruitment to the UASs. Subunits from the tail module, especially the so-called “Triade” Med2, Med3 and Med15, interact with specific TFs, such as Gal4, Gcn4 and Met4 at the UAS (reviewed in (Allen and Taatjes, 2015)). However, several works from the Morse’s group showed that the Tail is required for Mediator recruitment on a limited number of genes, mostly SAGA-dominated inducible genes.
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Several studies revealed indeed that transcription of some stress-responsive genes is dependent on the coordinated action of the Mediator tail, the SAGA complex and the Swi/Snf remodeler (Bhoite et al., 2001; Bryant and Ptashne, 2003; Govind et al., 2005; Herbig et al., 2010; Leroy et al., 2006).
However, Mediator recruitment on vast majority of genes and especially on TFIID-dominated house-keeping genes is instead Tail-independent (Ansari et al., 2012; Ansari and Morse, 2012, 2013).
Furthermore, the interactions between Mediator and TFs are not restricted to the Tail but involve also subunits of other modules such as Med17, Med7 and Med31 (Koschubs et al., 2009). It has also been shown that disruption of Mediator integrity in a Med17-temperature-sensitive allele does not affect binding of the tail module to UASs (Paul et al., 2015).
It is worth to mention that post translational modifications of some Mediator subunits (such as phosphorylation of Med13 and Med15) could contribute to Mediator interaction with TFs (reviewed in (Soutourina, 2018)). Finally, it has been shown that in vitro association of Mediator with chromatin is affected by the acetylated states of the histones (Liu and Myers, 2012; Zhu et al., 2011).
Mediator binding to the genome: a controversial topic
Though it was early well-established that disruption of Mediator affects transcription genome-wide, whether Mediator binds the UAS of all genes has been controversial matter of debate for long time.
While an early genome-wide ChIP-chip study of several subunits of Mediator detected Mediator binding on the UAS of essentially all yeast genes (Andrau et al., 2006), other similar studies could not detect any Mediator binding on many highly transcribed TFIID-dominated genes as RP genes (Fan et al., 2006). However, in 2009 the Morse group detected Mediator binding on RP gene promoters and showed that it is affected in a Rap1 temperature-sensitive mutant at non-permissive temperature (Ansari et al., 2009). A more recent ChIP-chip study on many Mediator subunits showed that Mediator binds to the UAS of many yeast genes but not to RP genes (Jeronimo and Robert, 2014).
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However, the same study showed that inhibition of Ser5 phosphorylation in Kin28 analog-sensitive strain stabilizes Mediator association with RNAPII and allows Mediator detection at core promoter also of RP genes. This observation suggests that the transient and highly dynamic interaction of Mediator with RNAPII makes particularly hard its detection on some of its target genes. In 2017 a technique alternative to ChIP-Seq named ChEC-Seq (Schmid et al., 2004; Zentner et al., 2015) was used to map Mediator binding genome-wide. This study revealed that Mediator binds the UAS of nearly all genes, including RP genes (Grunberg et al., 2016).