The Mediator Complex

THE MEDIATOR COMPLEX ¨ RKLUND* AND CLAES M. GUSTAFSSONÀ By STEFAN BJO

À

*Department of Medical Biochemistry, Umea˚ University, S-901 87 Umea˚, Sweden; Department of Medical Nutrition, Karolinska Institute, Novum, S-141 86 Huddinge, Sweden

I. Summary. .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . II. Saccharomyces cerevisiae Mediator. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . A. Identification of S. cerevisiae Mediator . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . B. Interactions with RNA Polymerase . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . C. Subunit Composition of Yeast Mediator. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . D. Global Gene Regulation. . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . III. Mediator Complexes in Higher Eukaryotes. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . A. Identification of Mammalian Mediator . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . B. Functional Studies of Metazoan Subunits . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . IV. Mechanism of Transcriptional Activation . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . A. Role of the RNA Polymerase II C-Terminal Domain . . . . . . . . . . . . . . . . . . . .. . . . . . . B. Structure–Function. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . V. Concluding Remarks . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . .

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I. Summary The Mediator complex acts as a bridge, conveying regulatory information from enhancers and other control elements to the general transcription machinery. The Mediator was originally identified in Saccharomyces cerevisiae and is required for the basal and regulated expression of nearly all RNA Pol II–dependent genes. Mediator complexes were recently identified also in metazoans, confirming a role for Mediator in transcription regulation in higher eukaryotes as well. In spite of its general significance for transcription control, the exact mechanisms of Mediator function remain unclear. We here review our understanding of the structure and possible models for the function of Mediator in yeast and metazoan cells.

II. Saccharomyces cerevisiae Mediator RNA polymerase II (RNA Pol II)–dependent transcription initiation supposedly proceeds in two stages. First there is a relief of repression by remodeling of chromatin structure at the promoter. This step is dependent on the activity of chromatin modifying or remodeling complexes (Urnov and Wolffe, 2001), which are recruited to specific promoters by regulatory proteins. Second, after remodeling of the promoter, a preinitiation complex containing RNA Pol II and the general transcription 43 ADVANCES IN PROTEIN CHEMISTRY, Vol. 67

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factors (GTFs) TFIIB, TFIID, TFIIE, TFIIF, and TFIIH is formed. At this stage, activators recruit GTFs and stimulate the assembly of the preinitiation complex on to the promoter. However, direct interactions between different activators and general transcription factors do not seem to be sufficient for transcription activation, as activators fail to stimulate transcription in systems reconstituted from pure RNA Pol II, basal factors, and purified template DNA.

A. Identification of S. cerevisiae Mediator In a search for a factor that could enable a response to transcriptional activators in a pure in vitro transcription system, R. D. Kornberg and colleagues isolated an activity from S. cerevisiae that was termed Mediator (Flanagan et al., 1991; Kelleher et al., 1990). The assay used was based on naked DNA templates and thus reflects the second stage of the transcription initiation process described above. The Mediator activity was purified to homogeneity and shown to be a holoenzyme form of RNA Pol II, made up of core 12-subunit RNA Pol II and a Mediator complex (Kim et al., 1994). Mediator was later also isolated as a discrete entity and identified as a multiprotein complex of 20 individual polypeptides (Table I; Myers et al., 1998). The functional activities identified for the Mediator were stimulation of basal transcription, support of activated transcription, and enhancement of phosphorylation of RNA Pol II by TFIIH kinase (Kim et al., 1994; Myers et al., 1998). Later studies also identified a histone acetyltransferase activity in the S. cerevisiae Mediator (Lorch et al., 2000).

B. Interactions with RNA Polymerase The C-terminal domain (CTD) of the largest subunit in RNA Pol II plays an important role in the function of Mediator (Myers and Kornberg, 2000). The domain, which consists of multiple heptapeptide repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser, is conserved in all eukaryotes studied to date. In S. cerevisiae, CTD truncations cause defects in transcriptional activation both in vivo and in vitro (Scafe et al., 1990). Two distinct forms of RNA pol II have been identified in S. cerevisiae. Most RNA Pol II molecules have an unphosphorylated CTD, but a portion of RNA Pol II molecules is highly phosphorylated. The unphosphorylated RNA Pol II associates with the promoter-bound initiation complex, whereas the phosphorylated form is responsible for active elongation (Cadena and Dahmus, 1987). The principal protein kinase involved in the phosphorylation of CTD has been identified as Kin28, a cyclin dependent kinase (cdk) and subunit of

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Table I Mediator Subunits in S. cerevisiae and Their Homologues in Saccharomyces pombe and Human Cells Saccharomyces cerevisiae Gene deletion phenotype

Protein mass (kD)

Nut1

Conditional

129

Gal11 Rgr1 Sin4 Srb4 Med1 Med2 Pgd1/Hrs1 Med4 Med6 Srb5 Med7 Med8 Rox3 Srb2 Nut2 Cse2 Srb7 Srb6 Med11 Srb8 Srb9 Srb10

Conditional Inviable Conditional Inviable Conditional Conditional Conditional Inviable Inviable Conditional Inviable Inviable Inviable Conditional Inviable Conditional Inviable Inviable Inviable Conditional Conditional Conditional

120 123 111 78 64 48 47 32 32 34 32 25 25 23 18 17 16 14 15 167 160 63

Srb11

Conditional

38

Subunit

Activity

S. pombe subunit

Human subunita

Pmc1

Med150

spSrb4

Med78

spMed4 spMed6

Med36 Med33

spMed7 spMed8 spRox3

Med34 Arc32b

spNut2

Med10

spSrb7 spSrb6

Med17

spSrb8

Med230

spSrb10

Cdk8

spSrb11

CyclinC

Histone acetyltransferase

Cyclin-dependent protein kinase Cyclin

a

We here use the nomenclature proposed by Rachez and Freeman (2001). The Arc32 protein has so far only been identified in the ARC complex (Naar et al., 1999). b

the general transcription factor TFIIH (Feaver et al., 1994). It is generally believed that Kin28-dependent phosphorylation of the CTD leads to a breakdown of the preinitiation complex and the transition from transcription initiation to elongation (Svejstrup et al., 1997). One of the cardinal activities of Mediator is its ability to stimulate phosphorylation of CTD by TFIIH (Kim et al., 1994; Myers et al., 1998). The level of stimulation can be more than 40-fold and is specific for the

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Kin28 kinase. The molecular basis for Mediator’s ability to stimulate CTD phosphorylation is unknown. However, the observation that Saccharomyces pombe Mediator is unable to stimulate TFIIH derived from S. cerevisiae indicates that specific interactions are formed between Mediator and TFIIH (Spahr et al., 2000).

C. Subunit Composition of Yeast Mediator The majority of genes encoding the yeast Mediator subunits had previously been identified in genetic screens for mutations affecting activation and repression of transcription. The presence of these well-characterized gene products in one single complex connected Mediator with a quartercentury of genetic analysis in yeast and at once established the relevance of Mediator function in vivo.

1. Srb Proteins As mentioned previously, CTD truncations cause defects in transcriptional activation both in vivo and in vitro (Scafe et al., 1990). The nine SRB genes were originally identified by R. A. Young and colleagues in a genetic screen for suppressors of RNA polymerase IIB, a version of the polymerase carrying only 11 instead of the normal 26–27 heptapeptide repeats in the CTD of the largest RNA Pol II subunit (Nonet and Young, 1989). The Srb proteins were later isolated in a complex with RNA Pol II, giving the first indication of the existence of an RNA Pol II holoenzyme (Koleske and Young, 1994; Thompson et al., 1993). Five of the SRB genes, SRB2, SRB4, SRB5, SRB6, and SRB7, encode core Mediator subunits, which are present in all Mediator preparations. Proteins Srb2, Srb4, Srb5, and Srb6 have been shown to interact in a subcomplex of Mediator together with Med6 and Rox3 (Lee and Kim, 1998). The Srb4, Srb6, and Srb7 proteins are all encoded by essential genes, and a temperature-sensitive(ts) mutation in the SRB4 gene shuts down nearly all RNA Pol II–dependent transcription at the nonpermissive temperature (Thompson and Young, 1995). SRB2 and SRB5 are nonessential genes with a slow growth phenotype (Nonet and Young, 1989; Thompson et al., 1993). SRB5 is needed for expression of genes involved in the pheromone response pathway, which is reflected in a defect in mating efficiency for srb5 cells (Holstege et al., 1998). A subgroup of Srb proteins (Srb8, Srb9, Srb10, and Srb11) forms a specific module that is present in holoenzyme preparations from cells growing exponentially in rich glucose medium, but is absent in stationary-phase

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cells (Hengartner et al., 1998). The SRB11 and SRB10 encode cyclin C and the cyclin C-dependent kinase, respectively (Liao et al., 1995). Genetic analysis indicates that the Srb8-11 module is involved in the negative regulation of a small subset of genes (Holstege et al., 1998). Srb8 is required for stable association of Srb10 and Srb11 with the holoenzyme inasmuch as holoenzyme preparations from Srb8 deletion strains lack Srb10 and Srb11 (Myer and Young, 1998). Homologues to Srb10 and Srb11 are found in some human Mediator preparations. Alleles of SRB8, SRB9, SRB10, and SRB11 have been identified as ssn (suppressors of snf1) mutations (Song et al., 1996). Two other genes encoding mediator subunits, ROX3 and SIN4, were also identified in the same genetic screen. The Snf1 kinase is a homologue of the mammalian AMP-activated protein kinase and is inactive in the presence of glucose (Woods et al., 1994). Snf1 functions by inactivating the Mig1 repressor, which binds to promoters of many glucose-repressed genes and recruits the Ssn6-Tup1 corepressor complex (Treitel and Carlson, 1995). Direct interactions have been demonstrated between Tup1 and the N-terminal domain of the Mediator subunit Srb7 (Gromoller and Lehming, 2000). Interestingly, Tup1 interaction with Srb7 precludes interaction between Srb7 and another Mediator subunit, Med6. The Srb7–Med6 contacts are believed to be part of a pathway that relays positive signals within Mediator, and the inhibition of this pathway could, therefore, explain the repressive activity of Tup1’s repressive activity. Cells lacking Snf1 cannot grow on any carbon source except glucose. Cells lacking both Snf1 and Mig1 can also grow on galactose and sucrose, but are still unable to grow on gluconeogenic carbon sources. Thus, some genes that are required for gluconeogenic growth are repressed by Mig1-independent mechanisms that operate downstream of Snf1. The SRB genes appear to also be involved in the Mig1-independent repression, as spontaneous mutations that allow snf1/mig1 cells to grow on gluconeogenic carbon sources have been identified in SRB8, SRB10, and SRB11 (Balciunas and Ronne, 1995). Even if the Srb8-11 module has mostly been implicated in negative regulation of transcription, it also appears to have a positive effect on some genes. When yeast cells enter stationary phase in response to certain types of nutrient limitations, there is a down-regulation of most RNA Pol II transcribed genes. However, the expression of some genes, such as YGP1, is induced under these conditions. In a genetic screen for mutants that are defective in the regulation of YGP1 expression (rye), Herman and colleagues showed that three of the RYE genes encode Srb9, Srb10, and Srb11 (Chang et al., 2001).

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2. Med Proteins The MED genes were grouped together because they were encoded by previously uncharacterized genes and their products were identified through peptide sequencing (Kim et al., 1994; Myers et al., 1998). Some of the Med proteins appear to be specific for S. cerevisiae, such as Med1, Med2, and Med11, whereas others like Med4, Med6, Med7, and Med8 are conserved in all Mediator-like complexes isolated to date (Table I). Deletion of MED1 causes a complex phenotype similar to mutations in SRB10 and SRB11, including suppression of snf1 (Balciunas et al., 1999). A MED1 deletion strain displays a partial loss of glucose repression and a slightly impaired induction of galactose-regulated genes. In contrast to many other Mediator subunits, the Med1 protein fused to the DNA-binding domain of LexA does not function as an activator in wild-type cells. However, LexA-Med1 fusion is a strong activator (400-fold) when expressed in an srb8, srb10, or srb11 deletion strain (Balciunas et al., 2003). It thus appears as if the Srb10-Srb11 cyclin kinase complex negatively regulates the function of Med1. Med2 forms a stable submodule with Hrs1/Pgd1 and Gal11 (Lee et al., 1999; Myers et al., 1999). A deletion of one of the genes encoding for these proteins will lead to a concomitant loss of all three proteins from the Mediator complex. Whole-genome analysis of MED2-dependent transcription indicates that the expression of about 200 genes is significantly decreased in med2 cells and, specifically, induction of several GAL genes was found to be defective in the med2 strain (Myers et al., 1999). However, it seems as it this Gal
phenotype is caused by a delay in induction rather than a reduction in galactokinase levels (Balciunas et al., 1999). Med4 is an essential gene with weak sequence homology to the human Mediator subunit Trap36 (Myers et al., 1998; Spahr et al., 2001). So far, no genetic studies involving Med4 have been presented. Med6 is an essential subunit with highly conserved homologues in all Mediator complexes studied to date (Lee et al., 1997). A ts mutation in S. cerevisiae MED6 showed defects in activation of several inducible promoters, but no effect on uninduced or constitutively expressed genes. The same pattern has also been observed for the Caenorhabditis elegans homologue, which is required for developmental stage-specific transcriptional regulation but dispensable for the expression of two constitutively expressed genes tested (Kwon et al., 1999). The effect on inducible yeast promoters is specific and coupled with certain classes of transcriptional activators. No effect was observed for GCN4-regulated genes, whereas both GAL4- and MAT1-regulated genes require Med6 for activation. However, deletion of MED6 does not affect the interaction between activators and

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Mediator, and thus points to a function of Med6 at a stage subsequent to recruitment of Mediator to promoters (Lee et al., 1999). S. cerevisiae cells lacking MED8 are inviable, but the function of Med8 is ambiguous (Myers et al., 1998). It was recently reported that Med8 binds directly to control elements in the invertase (SUC2) and hexokinase 2 (HXK2) promoters (Chaves et al., 1999). However, it is not clear whether this interaction involves the entire Mediator complex or merely the free Med8 protein. In S. pombe, Med8 has been identified as SEP15 in a genetic screen for mutants defective in cell separation (Zilahi et al., 2000). It is still possible that this effect is indirect, as the penetrance of the SEP15 mutation is incomplete in cell separation. Med11 is an essential gene required for MF1 transcription (Han et al., 1999).

3. Nut1 and Nut2 Proteins HO transcription is dependent on Swi4p and Swi6p for relief of repression by the URS2 region upstream of the HO promoter. NUT1 and NUT2 were, together with SIN4, ROX3, SRB8, SRB9, SRB10, and SRB11, originally isolated in a screen for mutants that would suppress the Swi4p/Swi6p dependence of a synthetic reporter gene containing part of URS2 (Tabtiang and Herskowitz, 1998). Nut1 appears to be specific to S. cerevisiae, whereas homologs to the essential Nut2 protein have been identified in all eukaryotic Mediator complexes isolated to date (Table I). Nut1 has been demonstrated to have histone acetyltransferase (HAT) activity, and purified Mediator can interact directly with free nucleosomes (Lorch et al., 2000). The exact role for the Nut1 HAT activity in Mediator function remains to be established.

4. Rox3 Protein The ROX3 gene, which is essential, was found in a search for mutants leading to overexpression of the heme-regulated CYC7 gene and was later also identified as SSN7 (Rosenblum-Vos et al., 1991; Song et al., 1996). ROX3 is also synonymous with RMR1, whose mutation can relieve glucose repression of the CYB2 gene (Brown et al., 1995). ROX3 does not only play a role in repression, as it is needed for full induction of the GAL1 gene in the presence of galactose (Brown et al., 1995).

5. Gal11, Sin4, and Rgr1 Proteins Gal11 was first described as an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes (Suzuki et al., 1988). It has also been implicated in enhancement of basal transcription (Sakurai et al., 1993), in negative regulation of the activity of the MCM1 transcription

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factor in Ty1 elements (Yu and Fassler, 1993), as an SNF gene (Vallier and Carlson, 1991), and as being involved in regulation of the structure and the position effect of telomeres (Suzuki and Nishizawa, 1994). However, the identification of Gal11 as a Mediator subunit has now led to a model in which Gal11 is part of a subcomplex of the Mediator that also includes Med2, Sin4, Pgd1, and possibly Nut1. This so-called Sin4 (or Gal11) module has been shown to be essential for the response to acidic transcriptional activator proteins such as Gal4VP16 (Lee et al., 1999; Myers et al., 1999). Gal11 interacts directly with the general transcription factors TFIIE and TFIIH (Sakurai et al., 1996), and a deletion of GAL11, MED2, or PGD1 causes synthetic lethality in combination with mutations in the large subunit of TFIIE. In addition, a ts mutation in KIN28, which encodes the kinase subunit of TFIIH, is lethal in a gal11 background (Sakurai and Fukasawa, 2000). RGR1 was isolated as a negative regulator of SUC2 (Sakai et al., 1990) but has also been identified as a negative regulator of the HO gene (Stillman et al., 1994). RGR1 is an essential gene, and an rgr1 strain shows pleiotropic effects such as resistance to glucose repression, ts lethality, sporulation deficiency in homozygous diploid cells, and abnormal cell morphology. SIN4, however, was identified as a negative regulator of GAL1 gene transcription, and it was also suggested that Sin4 alters chromatin structure in a way that affects transcriptional regulation ( Jiang and Stillman, 1992). However, several lines of evidence indicate that Rgr1 and Sin4 participate in the same regulatory pathways. RGR1 and SIN4 are negative transcriptional regulators of HO and IME1, and sin4 or rgr1 mutations have phenotypes similar to those caused by histone mutations, thus indicating that they act together in vivo to organize chromatin structure and to regulate transcription (Covitz et al., 1994; Jiang and Stillman, 1995; Stillman et al., 1994). These genetic interactions were confirmed biochemically in experiments in which the N-terminal domain of Rgr1 was shown to be important for the interaction between the Sin4 module and the rest of Mediator (Li et al., 1995).

6. Cse2 Protein Mutations in CSE1 and CSE2 lead to defects in chromosome segregation (CSE); (Xiao et al., 1993). CSE1 is an essential gene, whereas disruption of CSE2 causes chromosome missegregation, conditional lethality, and slow growth. Both Cse1 and Cse2 have been shown to interact physically with components of Mediator, using a high-throughput yeast two-hybrid system. Cse1 was shown to interact directly with Sin4, and Cse2 was identified as interacting directly with Med4 and indirectly, via Med4, to

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Srb7 and Med7. However, only Cse2 has been identified biochemically as a Mediator subunit (Gustafsson et al., 1998; Han et al., 1999). CSE2 encodes a 17-kDa protein that contains a putative leucine zipper motif, indicating that it may possess a DNA binding activity. CSE2 is required for Bas1/Bas2mediated basal transcription of amino acid biosynthetic genes, and holopolymerase isolated from cells lacking CSE2 display a 50% reduction in basal, nonregulated transcription (Han et al., 1999). On the basis of these results, it seems likely that the connection between CSE2 and chromosome segregation is indirect and involves transcription.

7. Soh1 Protein A human homologue of the yeast Soh1 protein has been identified as a subunit of the human mediator-like complexes TRAP, SMCC, and NC2 (Gu et al., 1999; Malik and Roeder, 2000), but Soh1 has not been demonstrated as a yeast Mediator subunit. The soh1, soh2, and soh4 mutants were isolated as suppressors of the temperature-dependent growth of the hyperrecombination mutant hpr1 (Fan et al., 1996). However, cloning of the corresponding genes indicates an involvement in RNA Pol II transcription. Soh2 is identical to the second-largest subunit of RNA Pol II, and Soh4 was identified as TFIIB. SOH1 encodes a novel 14-kD protein with limited sequence similarity to RNA polymerases. Mutations in SOH1 are synthetically lethal with mutations in RNA Pol II subunits and mutations in SUA7, which encodes yeast TFIIB.

D. Global Gene Regulation It is evident from the genetic characterization of Mediator that the general requirement for individual Mediator subunits in gene regulation will differ significantly. DNA microarray analysis of global gene expression supports this notion. Some Mediator components are needed for the regulated expression of nearly all genes, whereas others are only needed for a certain subset of genes (Holstege et al., 1998). The Srb4 ts strain demonstrates a decrease in the expression of 93% of all S. cerevisiae genes at the nonpermissive temperature. This value corresponds closely to that observed with a ts mutant in the largest subunit of RNA Pol II. In addition, the set of genes whose mRNAs are not significantly reduced in the RNA Pol II ts mutant exhibit the same behavior in the Srb4 ts experiment. The results indicate that genome-wide expression is as dependent on Srb4 as it is on core RNA Pol II, and that the Srb4-containing RNA Pol II holoenzyme is generally required for transcription. However, there are exceptions to the rule, and a small number of genes can indeed be expressed

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independently of Srb4, for example, CUP1 and SSN2 (Lee and Lis, 1998; McNeil et al., 1998). Interestingly, expression of the same subset of genes is also unaffected in cells lacking Kin28. This observation supports the notion that the function of Mediator is dependent of Kin28 and that regulation of TFIIH kinase activity is an essential part of Mediator’s ability to govern transcription in vivo. For most Mediator components, the effects on global gene expression are far less dramatic. Med6 is needed for expression of approximately 10% of all genes in the yeast genome, whereas about 16% of all genes are dependent on Srb5 function (Holstege et al., 1998). It should be noted that it is often difficult to distinguish between results that are a direct consequence of the loss of a specific Mediator subunit and those that are the result of a secondary effect. For example, global genome analysis has demonstrated a role for Med2 in the regulation of galactose inducible genes (Myers et al., 1999). However, as discussed above, Mediator purified from a med2 strain also lacks the Hrs1/Pgd1 and Gal11 proteins. Provided that the Hrs1/Pgd1 and Gal11 are also absent from Mediator in vivo, it would be impossible to distinguish the effects of a MED2 on global gene expression from the effects caused by deletion of HRS1/PGD1 and GAL11.

III. Mediator Complexes in Higher Eukaryotes Initially, it was unclear whether Mediator was specific for yeast or whether it had a counterpart in metazoan cells. The general view was that activators contacted TBP-associated factors, (TAFs), which in turn recruited TBP (TATA-binding protein) and subsequently other GTFs to specific promoters. This view was challenged by genetic studies in yeast, showing that TAFs are not required for transcriptional activation but, rather, contribute to the specificity of TBP-promoter interaction (Shen and Green, 1997). The first experimental indications of a metazoan Mediator complex came in 1996, when R. G. Roeder and coworkers isolated the multisubunit thyroid hormone receptor coactivator complex (TRAP), later identified as human Mediator (see Section III,A); (Fondell et al., 1996). The same year, R. A. Young and colleagues identified a human homologue to the Srb7 protein as a part of a larger RNA Pol II containing complex (Chao et al., 1996). Later, mammalian multiprotein complexes containing homologues of yeast Mediator proteins were identified in six laboratories (Boyer et al., 1998; Gu et al., 1999; Jiang et al., 1998; Naar et al., 1999; Rachez et al., 1999; Ryu et al., 1999; Sun et al., 1998). Similar to previous findings in yeast, human Mediator was shown to support activation in a fully reconstituted transcription system in the absence of TAFs (Oelgeschlager et al., 1998). These findings established Mediator as a major conduit of regulatory

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information from regulatory DNA elements to promoters across the whole range of eukaryotes, from yeast to man.

A. Identification of Mammalian Mediator The general importance of Mediator for both activated and repressed transcription in mammalian cells is also reflected in the multitude of ways the human Mediator complex was identified. The TRAP defined on p. 14 was purified by Roeder and coworkers as a complex associated with human thyroid hormone receptor alpha purified from HeLa cells grown in the presence of thyroid hormone (T3; Fondell et al., 1996). TRAP could also support activation of transcription in vitro from a promoter template containing T3-response elements. In parallel, the same laboratory used HeLa-derived cell lines expressing epitope-tagged hSrb7, hSrb10, or hSrb11 to identify a similar complex called SRB/MED-containing cofactor complex (SMCC; Gu et al., 1999). TRAP and SMCC have been shown to be identical (Ito et al., 1999). The DRIP (Vitamin D3 receptor [VDR] interacting proteins) complex was purified using a VDR ligand-binding domain affinity matrix (Rachez et al., 1998). DRIP is needed for full transcriptional activity of VDR on naked DNA templates in vitro. Another complex, ARC (activator-recruited cofactor), was identified as a complex that enhances transcription activation by SREBP-la, VP16, and the p65 subunit of NF-kappaB using chromatin-assembled DNA templates (Naar et al., 1999). Characterization of the subunits of DRIP and ARC showed that the two complexes are highly related—if not identical—to each other and also to the TRAP/SMCC complexes (Rachez et al., 1999). A role for Mediator in the transcription activation program that is initiated by viral infections of mammalian cells was revealed when Berk and colleagues identified a human homologue to the C. elegans Sur-2 protein as an in vivo target for the adenovirus E1A protein (Boyer et al., 1999). Further purification identified Sur-2 as a member of a mulitprotein complex containing human homologues to yeast Mediator proteins. This human Mediator could also support activation by Gal4-E1A as well as Gal4VP16 in a defined in vitro transcription system. TRAP/SMCC, DRIP, ARC, and human Mediator are virtually identical in their subunit composition (Malik and Roeder, 2000). Another set of Mediator-like complexes has also been isolated that appear to correspond to a submodule of the larger Mediator. This has led to speculations that the Mediator might exist in two separate forms. One of these smaller complexes is PC2, a component of the coactivator fraction USA (Malik et al.,

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2000; Meisterernst et al., 1991). PC2 can support activated transcription in vitro, but only in the presence of two other cofactors, PC3/topoisomerase I and PC4. Two other small Mediator complexes, CRSP (cofactor required for Sp1) and mouse Mediator, were both identified using a biochemical fractionation ( Jiang et al., 1998; Ryu et al., 1999). Interestingly, CRSP and PC2 could only support activation in the presence of TAFs (Malik et al., 2000; Ryu et al., 1999), This could indicate a yet-to-be-defined functional relationship between TAFs and the smaller form of Mediator. Another small Mediator complex is NAT (negative regulator of activated transcription). In contrast to the other Mediator complexes, NAT displayed an inhibitory effect on transcription in vitro (Sun et al., 1998). It is unclear whether the complexes described above represent distinct functional entities or whether the differences that exist between them are consequences of different purification methods. This was recently studied in experiments in which HeLa cell nuclear extracts were resolved directly by gel filtration (Wang et al., 2001). Only one peak of human Mediator, stoichiometric to the levels of GTFs in HeLa cells, was detected and revealed that the human Mediator had a molecular weight of about 2 MDa. This indicates that the smaller-sized complexes (CRSP, mouse mediator, PC2, and NAT) are either subcomplexes of the larger complexes (TRAP/SMCC, NAT, DRIP, ARC, and human Mediator) formed by dissociation of the larger complexes during fractionation or are much less abundant. It was also found that the 2-MDa human mediator is present in two forms of indistinguishable size, one containing and one lacking the Srb10/Srb11 CDK-cyclin pair. Recently, a smaller form of the Mediator was also identified in S. cerevisiae nuclear extracts and termed Medc (Liu et al., 2000). This complex contains all the subunits of Mediator with the exception of Rgr1, Rox3, Nut1, and the Sin4 module. Medc is less abundant than Mediator and is also less active in transcription. The functional role of Medc remains to be established, but it could lend biochemical support for the existence of two forms of the Mediator complex not only in higher eukaryotes but also in yeast.

B. Functional Studies of Metazoan Subunits The function of individual metazoan Mediator subunits has been studied by different methods; that is, by RNA interference (RNAi) and chemical mutagenesis in C. elegans, P insertions in Drosophila, homologous recombination in mouse, and studying spontaneous mutations in human cells (Ito et al., 2000; Kwon et al., 1999; Nilsson et al., 2000; Philibert et al., 1998; Singh and Han, 1995; Spradling et al., 1999; Tudor et al., 1999; Zhu et al., 2000).

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RNAi experiments in C. elegans showed that Med6, Med7, and Nut2 are essential for embryogenesis (Kwon et al., 1999). Interestingly, Med6, Med7, and Nut2 were found to be required for expression of two developmentally regulated genes but dispensable for expression of two ubiquitously expressed genes. RNAi experiments also show that the C. elegans homolog of Rgr1 is required early in embryogenesis (S. Tuck, personal communication). In contrast, worms homozygous for putative null mutations in the C. elegans Med130/sur-2 are viable and fertile (Singh and Han, 1995), as are worms with reducedMed230/sop-1 activity (Zhang and Emmons, 2000). sop-1(RNAi) worms are also viable, but it is not known whether such worms completely lack sop-1 activity. The sur-2 gene product appears to have a role in multiple developmental stages, operating downstream of Ras and MAP Kinase (Nilsson et al., 2000; Singh and Han, 1995). Animals with reduced sop-1 activity can bypass the requirement for PAL-1 (a homeobox protein) for neurogenesis in the male tail (Zhang and Emmons, 2000). In wild-type animals, sop-1 is thought to be a repressor of Wnt signaling. Two Mediator subunits, Med220/TRAP220 and Srb7, have been studied via inactivation of the corresponding genes in mice. In the Srb7 study, heterozygous ES cells and animals showed no phenotype (Tudor et al., 1999). However, no homozygous ES cells could be obtained, and homozygous embryos were only found up to the blastocyst stage, thus indicating that the Srb7 gene product is essential for both embryonic development and cell viability. Trap220 þ/
mice were fertile and phenotypically normal except for being slightly smaller compared with their Trap220 þ/þ littermates as a result of pituitary hypothyroidism (Ito et al., 2000; Zhu et al., 2000). However, Trap220
/
embryos died at embryonic day 11 with defects in development of the central nervous system, cardiac and large vessel enlargement, and defects in placental vasculature. Finally, in Drosophila melanogaster, a mutation generated by P insertion demonstrated an essential function also for the Med78 subunit (Spradling et al., 1999).

IV. Mechanism of Transcriptional Activation The molecular mechanism for Mediator-dependent transcriptional activation is still not completely understood. Specific interactions have been demonstrated between various activators and Mediator subunits in both S. cerevisiae and mammalian Mediator. In yeast, the VP16, Gal4, and Gcn4 proteins all interact directly with Gal11, and in the case of Gcn4, additional contacts are made with Hrs1/Pgd1 (Park et al., 2000). Specific interactions have also been reported between the Gal4 activation domain

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and Srb4 (Koh et al., 1998). Direct interactions with mammalian Mediator have been demonstrated for a number of nuclear hormone receptors. These include TR, VDR, retinoic acid receptor  (RAR), retinoic X receptor , peroxisome proliferator-activated receptor , proliferatoractivated receptor , estrogen receptor , and glucocorticoid receptor (Hittelman et al., 1999; Yuan et al., 1998; Zhu et al., 1997). Many of the receptors seem to interact with two closely located nuclear receptor (NR) interaction boxes (LXXLL motifs) in the Med220/TRAP220 subunit of mammalian Mediator. A number of other interacting partners (i.e., p160, p300CBP, pCAF/SAGA, and SWI/SNF) have also been identified for the NR family (Aalfs and Kingston, 2000; Lemon and Freedman, 1999). All these coactivator complexes possess chromatin modifying or remodelling activities, whereas Mediator is supposed to operate mainly on the basal transcription machinery. A model has been proposed in which unliganded NRs initially function by binding to their target sites in chromatin in complex with different corepressor complexes. Binding of ligand to promoter-bound NRs leads to an exchange of NR-bound factors from corepressors to chromatin-remodeling coactivators. The remodeling of the promoter sequence surrounding the NR binding site then leads to recruitment of Mediator and subsequent or concomitant formation of a functional preinitiation complex. Given the specific interactions described between activators and Mediator, as well as the gene-specific effects observed for individual Mediator subunits, it seems likely that recruitment of RNA Pol II to the preinitiation complex plays an important role for Mediator function. This notion is supported by the finding in yeast that LexA-fusions to many individual Mediator subunits strongly activate transcription from a reporter containing LexA-binding sites 50 to the promoter (Balciunas et al., 1999; Song et al., 1996). However, the recruitment model does not take into account the specific genetic, functional, and physical interactions demonstrated between Mediator and CTD. A model to explain these interactions has been proposed (Svejstrup et al., 1997). It is based on the observation that RNA Pol II engaged in active transcription lacks associated Mediator. Formation of the preinitiation complex is dependent on the holoenzyme form of RNA Pol II, but Mediator is then released at the end of initiation or early in RNA chain elongation, as shown by its absence from the transcribing polymerase. During the initiation of transcription, Mediator stimulates CTD phosphorylation by TFIIH. Because Mediator is unable to bind to the hyperphosphorylated form of RNA Pol II, this eventually leads to dissociation of RNA Pol II from the preinitiation complex as transcriptional elongation begins. After completing a round of transcription, the CTD is dephosphorylated by

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a CTD-phosphatase. The unphosphorylated RNA Pol II can finally enter a new cycle of transcription by interacting with Mediator. This model recently gained support from results obtained by Hahn and collaborators (Yudkovsky et al., 2000). Using an immobilized template assay, they demonstrated that Mediator remains at the promoter after transcriptional initiation (Yudkovsky et al., 2000). Mediator forms a scaffold together with TFIID, TFIIA, TFIIH, and TFIIE that facilitates reinitiation of transcription from the promoter. Interestingly, the scaffold is stabilized in the presence of certain activators, for example, Gal4-VP16, immediately suggesting one possible mechanism for Mediator-dependent transcriptional activation. The question of how RNA Pol II is recruited to a promoter on activation was recently addressed in two independent systems (Cosma et al., 2001; Park et al., 2001). In the first paper, the ordered binding of factors to the HO promoter was studied using chromatin immunoprecipitation. Activation of HO is initiated in late mitosis by inactivation of the Cdk1 kinase via anaphase-promoting complex–mediated proteolysis of its B-type cyclin partners. This leads to translocation of the Swi5 transcription factor from the cytoplasm to the nucleus where it recruits the Swi/Snf chromatinremodeling complex to the HO promoter. The promoter-bound Swi/Snf then recruits the SAGA HAT complex to the promoter. Remodeling of the HO promoter permits binding of the transcriptional activator SBF, which is essential for activation of HO. Although these initial steps were well described previously, the function of SBF in the final steps of HO-activation has been unclear. However, the chromatin immunoprecipitation experiments clearly show that SBF functions in two steps. First, SBF recruits Mediator, but not RNA Pol II or GTFs, to the promoter by a mechanism that is independent of Cdk1. Activation of HO by recruitment of RNA Pol II and GTFs does not occur until the G1 phase of the cell cycle, when Cdk1 is activated by binding to the G1 cyclins. The target for Cdk1 in this process in so far unknown. A similar stepwise mechanism for transcriptional activation has also been proposed on the basis of studies of recruitment of the transcriptional activator HSF, Mediator, and RNA Pol II to the heat shock promoter of Drosophila polythene chromosomes (Park et al., 2001). Using different techniques, it was observed that on heat shock, both HSF and Mediator are rapidly recruited to the hsp70 promoter in a manner that is independent of the presence of a core promoter. This recruitment was not accompanied with a corresponding increase in RNA Pol II or GTFs and was also independent of the presence of the RNA Pol II inhibitor -amanitine. The results above are in line with the results in yeast discussed earlier, demonstrating that recruitment of holoenzyme is not needed for each round of transcription (Yudkovsky et al., 2000). Rather, the

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Mediator–RNA Pol II interaction is dynamic, and both Mediator and several GTFs remain at the promoter after release of the polymerase and can function as a scaffold for reinitiation by polymerase devoid of Mediator and GTFs.

A. Role of the RNA Polymerase II C-Terminal Domain The role of CTD for metazoan Mediator is still controversial. The S. cerevisiae Mediator interacts directly with CTD and needs CTD to stimulate basal transcription and support transcriptional activation (Myers et al., 1998). In contrast, in vitro studies of human Mediator have demonstrated activated transcription using a CTD-less polymerase (Gu et al., 1999). The molecular basis for the observed differences remains unclear. However, even if direct interactions have been demonstrated between CTD and Mediator, structural studies indicate that the most pronounced contacts are CTD independent (Asturias et al., 1999). It is therefore possible that Mediator may recruit RNA Pol II in a CTD independent fashion in vitro. RNA Pol II used by Mediator in the cell will undoubtedly contain an intact CTD. So what is then the molecular function of CTD? We favor a model in which the major role of CTD is to break the interaction between Mediator and RNA Pol II on CTD hyper-phosphorylation rather than being essential for formation of a Mediator-RNA Pol II complex. In support of this view, Reinberg and collaborators have demonstrated preferential binding of the human Mediator (NAT complex) to unphosphorylated CTD over phosphorylated CTD (Sun et al., 1998). In this respect it could be of interest to investigate the properties of Mediator isolated from Srb mutant strains, which suppress the mutant phenotypes of a truncated CTD in vivo. Perhaps the srb mutants weaken the CTD independent interactions formed between Mediator and RNA Pol II. This could facilitate dissociation of RNA Pol II and Mediator on CTD-hyperphosphorylation, and thus suppress the functional consequences of a truncated CTD. In support of this view, structural and biochemical studies indicate that the Srb2, Srb4, Srb5, and Srb6 proteins are located in the head domain of Mediator—the domain responsible for CTD-independent interactions with RNA Pol II (see following).

B. Structure–Function The subunit composition of the S. cerevisiae Mediator is clearly distinct from similar complexes found in other eukaryotic cells. Only eight out of 20 core Mediator subunits have a highly homologous counterpart in

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mouse and human Mediator (Table I). The low degree of conservation at the primary sequence level has evoked the idea that the metazoan Mediator is significantly different both in structure and function from yeast Mediator. Another possible interpretation is that Mediator functions as an interface between rapidly evolving gene-specific regulatory proteins and the highly conserved basal transcription machinery. According to this view, the conserved Mediator core of only eight proteins found in all eukaryotic cells is responsible for contacts with the basal transcription machinery. Subunits responsible for interactions with gene specific activators and repressors will be less conserved. In this view, the subunit composition may vary between eukaryotic cell types but the basic mechanisms of Mediator-dependent transcriptional regulation are the same. In support of this model, one can note that only essential gene products are conserved between the Mediator complex in yeast and higher eukaryotes (Table I). Nonessential gene products appear to be species specific. In fact, this is also true if one compares Mediator complex isolated from S. cerevisiae with the corresponding complex from fission yeast, S. pombe. The two species were separated early in evolution, and the 10 subunits conserved between them are all encoded by essential S. cerevisiae genes (Spahr et al., 2001). Support for the existence of a conserved Mediator core comes also from structural studies. Single-particle analysis by electron microscopy has demonstrated striking structural similarities between Mediator isolated from yeast, mouse, and human cells (Asturias et al., 1999; Dotson et al., 2000). In two-dimensional projections, the isolated Mediator purified from S. cerevisiae and mouse cells appears compact. When RNA Pol II is present, however, these Mediators adopt an extended conformation and embrace the globular Pol II. The extended structure reveals three distinct submodules of Mediator: a head, a middle, and a tail region. Direct contacts are formed between RNA Pol II and the head and middle region. The largest part of Mediator is made up of an elongated tail region, which does not appear to contact the RNA Pol II.

1. Tail Region ScMediator isolated from a sin4 strain lacks the Sin4 protein as well as Gal11, Med2, and Hrs1/Pgd1 (Myers et al., 1999). As mentioned previously, these proteins, the Sin4 module, are needed for the function of a wide variety of activators, including Gal4 and Gcn4. The module does not, however, appear to be important for other Mediator functions such as stimulation of basal transcription or CTD phosphorylation. The Sin4 module corresponds to the tail region, as image reconstruction of the
sin4 Mediator lacks this part of Mediator (Dotson et al., 2000). In vivo

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and in vitro evidence thus indicates that this region plays an important role for activator and repressor interactions with Mediator. The Sin4 module is known to interact with the rest of the Mediator complex through the terminal domain of the Rgr1/TRAP170 subunit, because deletion of this domain causes the loss of the entire module (Li et al., 1995). These observations, together with the fact that head and tail domains do not interact in the extended conformation of Mediator (Asturias et al., 1999), indicate that Rgr1/TRAP170 constitutes the part of the middle domain that is located most proximal to the tail domain.

2. Middle Region Biochemical analysis has identified two stable subcomplexes within Mediator (Lee and Kim, 1998). One of these subcomplexes (the Rgr1 module) contains Rgr1 together with Med1, Med4, Med7, Med8, Srb7, and probably Nut2. Many of the subunits of the Rgr1 submodule have a conserved homolog in S. pombe and metazoan Mediator complexes (Table I). These proteins include Med7, Nut2, and Srb7, which have recently been shown experimentally to form a stable complex when coexpressed from recombinant baculoviruses in insect cells (Han et al., 2001). On the basis of the structure of the
sin4 Mediator, it appears likely that the middle region corresponds to the Rgr1 module. The electron microscopy structures indicate a possible direct contact between RNA Pol II CTD and this middle region (Asturias et al., 1999).

3. Head Region By way of exclusion, the proteins not associated with the tail or middle region of the complex will probably correspond to the head region. These proteins correspond to second stable subcomplex of scMediator identified by biochemical analysis, the Srb4 module, which is composed of the Med6, Rox3, Srb2, Srb4, Srb5, and Srb6 proteins (Lee and Kim, 1998). Support for this module comes from the observation that the Med6, Srb2, Srb4, Srb5, and Srb6 proteins can form a stable complex on coexpression in insect cells (Lee et al., 1998). In fact, the Srb2 and Srb5 appear to form a subcomplex of the module, as Mediator purified from
srb2 cells also lack the Srb5 subunit (S. Bjo¨rklund, unpublished observations). Genetics experiments also support the postulated module, as Med6 and Srb6 both have been identified as dominant suppressors of a ts mutation in SRB4 (Lee et al., 1998). EM structures show that the most pronounced contacts between Mediator and RNA Pol II take place in the head region, which seems to interact with a part of RNA Pol II different from the CTD (Asturias et al., 1999). In fact, Med6 copurify with the core RNA Pol II rather than with the rest of

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Fig. 1. The yeast RNA Polymerase II holoenzyme revealed by electron microscopy and image processing. (Asturias et al., 1999). (A) The extended Mediator contains three distinguishable regions; head (h), middle (m), and tail (t). The globular density embraced by Mediator is identified as RNA polymerase II. The outline of a projection of the previously determined polymerase three-dimensional structure is superimposed (dark line), with the point of attachment of the C-terminal domain (dark circle) and the location of the DNA-binding channel (c) indicated. (B) Tentative subunit organization for the holoenzyme. The model is based on available structural information and reported physical interactions. The surface of each subunit has been calculated by assuming a globular shape and drawn in scale. Subunits in red have reported homologs in Saccharomyces pombe and, with the exception of Rox3 and Srb6, also in mammalian Mediator. The yellow subunits are specific for Saccharomyces cerevisiae.

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the Mediator in extracts from certain yeast strains (Balciunas et al., 1999). As expected, many of the proteins of the head region are also conserved in other species, including homologs to Med6 and Srb4 in mammalian Mediator complexes (Table I). In summary, we would like to propose an onion-like structure of Mediator (Fig. 1). It appears as if the head and middle regions of Mediator contain all the conserved and essential subunits of the S. cerevisiae complex. In concordance with the core Mediator model, these are also the polymerase-interacting regions, and it is likely that the conserved subunits of these domains are faced toward the RNA Pol II. In contrast, nonconserved subunits in the head and middle domains and all subunits in the tail region interact with activators, and repressors are facing outward to receive signals from regulatory proteins. In agreement with this, structural comparison between S. cerevisiae and human Mediator has demonstrated striking structural similarities in the head and middle regions (Asturias et al., 1999; Dotson et al., 2000). The tail region of the human complex is large and differs significantly in its structure from S. cerevisiae. One could speculate that this region of the human Mediator complex may contain a number of large and metazoan-specific subunits involved in activator and repressor interactions; for example, TRAP220, TRAP230, and TRAP240 (Malik and Roeder, 2000).

V. Concluding Remarks The discovery of Mediator has changed our view of transcriptional regulation. This multiprotein complex is now established as the main transducer of regulator information from enhancers and other control elements to the promoter. Mediator seems to form an interface between gene-specific regulatory proteins and the highly conserved basal transcription machinery. A conserved core of only eight proteins found in all eukaryotic cells is responsible for contacts formed with RNA Pol II and TFIIH. Other, species-specific subunits are mainly responsible for direct interactions with regulatory proteins. The subunit composition of Mediator may therefore vary between different eukaryotic cell types, but the mechanisms of Mediator-dependent transcriptional regulation are highly conserved.

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