InteractiveFly: GeneBrief

Cyclin-dependent kinase 8: Biological Overview | References

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. This study investigated the functions of the four Cdk8 module subunits in Drosophila. Physical interactions detected among the four fly subunits provide support for a structurally conserved Cdk8 module. The in vivo functions of this were analyzed 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 (Loncle, 2007).

Cell fate specification during development is ensured by the progressive deployment of a great variety of DNA-bound transcription factors that control gene expression. Much of the specificity of this process occurs at the pre-initiation step of transcription. There, an evolutionarily conserved complex of ~25 subunits termed Mediator (MED) plays a pivotal role in the fine-tuned recruitment of the general RNA polymerase II (PolII) initiation machinery to gene promoters (Kornberg, 2005). Indeed, it is now widely accepted that MED integrates and conveys regulatory signals by bridging specific activators and repressors to PolII and associated general transcription factors (GTFs) (Bjorklund, 2005 Conaway, 2005 Kim, 2005; Malik, 2005; Loncle, 2007 and references therein).

The structural and functional organization of MED has been well characterized in the budding yeast Saccharomyces cerevisiae (Bjorklund, 2005). It is composed of three core modules that can interact with an additional, separable regulatory module consisting of a cyclin-dependent kinase (Cdk), Cdk8, its C-type cyclin (CycC) partner, Med12 and Med13 (Borggrefe, 2002). This interaction appears to be transient, since a recent genome-wide analysis of MED subunit localization on chromosomal DNA revealed that the Cdk8 module occupies the same sites as core MED but at generally lower levels (Andrau, 2006). The four yeast Cdk8 module subunits display similar loss-of-function phenotypes that reflect their shared specific requirements for cellular adaptation to environmental stresses, notably nutrient deprivation and heat shock (Carlson, 1997). A CDK8 missense mutant that inactivates the kinase activity without affecting its incorporation into MED provokes the same defects as a deletion allele (Liao, 1995, Borggrefe, 2002), and affects the transcription of the same gene subset (Holstege, 1998). It has therefore been proposed that all functions of the four Cdk8 module components are mediated by the kinase activity of Cdk8 (Myer, 1998). A comparative genome-wide analysis revealed that each of the four subunits regulates essentially the same genes (van de Peppel, 2005). Although most target genes are subjected to repression, some are upregulated by Cdk8 module activity. The regulatory function of Cdk8 kinase involves phosphorylations of the carboxy-terminal domain (CTD) of the large PolII subunit, other MED subunits, GTFs and gene-specific activators (Hengartner, 1998; Hirst, 1999; Chi, 2001; Vincent, 2001; Nelson, 2003; Hallberg, 2004; Liu, 2004; van de Peppel, 2005). Both reconstituted Cdk8-CycC pair and purified Cdk8 module can phosphorylate the CTD in vitro (Liao, 1995; Borggrefe, 2002). Thus, yeast Med12 and Med13 are apparently not essential for Cdk8 catalytic activity, raising questions about their precise roles within the Cdk8 module (Loncle, 2007 and references therein).

All four subunits of the S. cerevisiae Cdk8 module have structural counterparts in the distantly related fission yeast Schizosaccharomyces pombe and in metazoans, suggesting that the architecture of this regulatory MED module has been conserved during evolution. Direct support for this hypothesis is provided by the isolation of functionally distinct S. pombe and mammalian MED forms simultaneously harboring, or lacking, Cdk8, CycC, Med12 and Med13 (Conaway, 2005; Malik, 2005). Consistent with a conserved repressive role for the Cdk8 module, purified mammalian MED that contains Cdk8 harbors little or no PolII, whereas isolated core complexes are associated with near stoichiometric amounts of PolII subunits (Sato, 2004; Malik, 2005). Thus, metazoan Cdk8 module may inhibit stable PolII-MED interaction, as in yeasts (Hengartner, 1998; Elmlund, 2006). Finally, Med12 depletion in human cells results in reduced levels of Cdk8 protein (Kim, 2006), in the cell and within MED (Loncle, 2007 and references therein).

The physiological roles of Cdk8 module subunits in eukaryotes other than fungi have only recently begun to come to light. In the slime mold Dictyostelium discoideum, both Cdk8 and Med13 mutants are unable to form multicellular aggregates upon nutrient deprivation . In the worm Caenorhabditis elegans, Med12 and Med13 mutants show similar defects of the female vulva and male tail. Finally, Drosophila melanogaster Med12 and Med13 have indistinguishable loss-of-function phenotypes in eye and wing morphogenesis (Treisman, 2001; Janody, 2003). These genetic studies collectively suggest that metazoan Med12 and Med13 act together within a Cdk8 module. However, a comparative functional analysis of Cdk8, CycC, Med12 and Med13 has not yet been performed in any metazoan (Loncle, 2007).

Drosophila represents an appropriate genetic model to examine, in vivo, the functional relationships of the four Cdk8 module components in a higher eukaryote. This study found that Drosophila Cdk8 and CycC can physically interact with Med12 and Med13, reinforcing the idea that these conserved subunits retain a Cdk8 module architecture from yeast to metazoans. To examine the developmental roles of the fly Cdk8 module subunits, null alleles of Cdk8 and CycC were generated and their effects were compared with previously described loss-of-function alleles of Med12 and Med13. All four genes are essential for the development of the organism but not for cell viability. Consistent with a paired action of Cdk8 and CycC in vivo, mosaic adults harboring clones of Cdk8^- or CycC^- cells exhibit indistinguishable defects in leg, eye and notum differentiation. However, although the mutant phenotypes for Cdk8-CycC closely resemble those for Med12-Med13 in some situations, they diverge significantly in others. These effects on adult morphology are corroborated at the level of gene expression for several developmentally important genes, including decapentaplegic, dachshund, bric-à-brac-2 and senseless, whose expression patterns are differentially affected, according both to the tissue and the mutated subunit. These results reveal that Med12 and Med13 can have specific roles distinct from cyclin-regulated Cdk8 activity and thus underline the functional diversity of Cdk8 module subunits during development (Loncle, 2007).

Drosophila Cdk8 and CycC have been shown to interact both in vitro and in vivo (Leclerc, 1996). Similarly, based on their identical mutant phenotypes and co-immunoprecipitation from extracts of embryos overexpressing both proteins, Drosophila Med12 and Med13 have been proposed to function as a unit in vivo (Treisman, 2001; Janody, 2003). To examine the possibility of direct binding between fly Med12 and Med13 with Cdk8 and CycC, the glutathione S-transferase (GST) pull-down assay was used. GST-Cdk8 and GST-CycC fusion proteins were expressed in bacteria and purified from total cell extracts by affinity on glutathione beads. Under relatively stringent conditions, GST-CycC specifically interacted with in vitro translated ³⁵S-labeled Cdk8, as previously shown (Leclerc, 1996). In addition, GST-Cdk8 bound to radiolabeled Med12 as well as Med13, whereas GST-CycC did not. However, under slightly less stringent conditions, GST-CycC also interacted with Med12 and Med13 (Loncle, 2007).

To examine the developmental functions of the Drosophila Cdk8 module, mutants for each subunit were compared. Although null alleles of Drosophila Med12 and Med13 have been described (Treisman, 2001; Janody, 2003), no mutants for Cdk8 or CycC were known. Imprecise excision of nearby P transposons were used to generate Cdk8 and CycC loss-of-function mutations. Cdk8 alleles were generated by excising a homozygous viable P-element insertion situated 328 base pairs (bp) from the 3' end of Cdk8 mRNA, in the 5'-untranslated region (UTR) of the neighboring I-2 gene. One recessive lethal allele chosen for subsequent analyses, Cdk8^K185, retains the P extremity in I-2 and deletes 882 bp, including the C-terminal one-third of Cdk8 protein-coding sequences. This allele causes lethality in late third-instar larvae (L3) and behaves as a null in complementation test with a larger deletion. Further, homozygotes for Cdk8^K185 are fully rescued to yield viable, morphologically normal fertile adults by a Ub-Cdk8 transgene that ubiquitously expresses normal Cdk8 protein. Finally, wild-type Cdk8 protein was not detected in Western analyses of ventral nerve cords and associated imaginal discs prepared from mutant L3 tissues (Loncle, 2007).

To obtain loss-of-function CycC alleles, imprecise excisions of a viable insertion were generated in its 5'-UTR region. One recessive lethal allele, CycC^Y5, deletes the entire P insert plus 1429 and 1304 bp of 5'- and 3'-flanking genomic DNA, respectively. Thus, CycC^Y5 removes all CycC protein-coding sequences plus parts of its overlapping 5'- and 3'-gene neighbors, CG3731 and CG3641. CycC^Y5 is lethal at early pupal stage and, consistently with molecular data, behaves as a null allele based on complementation test with a larger chromosomal deficiency. CycC^Y5 homozygotes could be rescued to a limited extent through a combination of arm-GAL4 and UAS-CycC transgenes (arm>CycC) that directs ubiquitous CycC expression, although emerging adults showed some loss of sensory organs and malformed sex combs. Adding a second arm>CycC copy led to extensive rescue, and such adults were morphologically normal. Finally, no CycC protein was detected in Western blot analyses from mutant L3 tissues. It is concluded that Cdk8^K185 and CycC^Y5 are null alleles whose effects are solely due to the loss of Cdk8 and CycC activity, and they are referred to as Cdk8^- and CycC^- (Loncle, 2007).

The developmental effects of these new Cdk8 and CycC mutants were then compared with those of the characterized null alleles Med12^T241 and Med13^T606 (Treisman, 2001). All four mutants are recessive lethal, showing that each Cdk8 module component is required for the viability of the organism. However, Med12 and Med13 homozygous animals die as late embryos/early first-instar larvae, whereas Cdk8^- and CycC^- mutants survive until late L3/early pupae. Given that mRNAs encoding each Cdk8 module subunit are maternally contributed to the embryo, this discrepancy might reflect their differential quantities and/or perdurance. Alternatively, Cdk8 module subunits might be required for divergent developmental processes. To avoid the complicating effects of maternal pools which partially compensate for zygotic mutations in early development, mitotic recombination was used to generate clones of homozygous mutant at later stages, where normal proteins are no longer detected. Clones of Cdk8^- or CycC^- cells were readily detected in all examined L3 imaginal discs. This indicates that neither gene is required for cell viability, as is also the case for Med12 and Med13 (Boube, 2000 Treisman, 2001). Taken together, these results show that each Cdk8 module subunit is required for the development of the organism, but is dispensable for cell viability (Loncle, 2007).

To compare functions of the four Cdk8 module components in adult development, mosaic animals were generated harboring clones of Cdk8^-, CycC^-, Med12^- or Med13^- cells. In light of a previously described role for Med13 in attributing sex comb cell identity (Boube, 2000), mutant clones were targeted to distal leg imaginal discs by expressing the Flp recombinase under the control of Distal-less (Dll) regulatory sequences (Dll>Flp). In initial essays, the 'Minute' technique of growth enhancement was used to generate large mutant clones covering the majority of the imaginal discs by the end of larval development. The resulting adult Legs were severely stunted, but were less affected for Cdk8^- and CycC^- than for Med12^- and Med13^-. As the severity of the phenotypes rendered interpretation difficult, these experiments were repeated without growth enhancement. In these conditions, numerous mutant clones were systematically observed in larval leg discs, and resulted in tarsal segmentation defects in all three pairs of adult legs for each of the four subunits (Loncle, 2007).

The effects of Cdk8 module mutants on male prothoracic (T1) distal legs were documented. The basitarsus of a wild-type male T1 leg normally displays a sex comb composed of an aligned row of about 11 thick and darkly pigmented bristles, or teeth. All four single mutants led to sex comb defects at similar frequencies (63%-85%). However, two distinct classes of defects could be discerned. For Cdk8^- and CycC^- clones, the mean number of sex comb teeth was slightly elevated (12 and 13, respectively). However, nearly half of the mutant samples also presented at least three discontinuities in the normal tooth alignment, leading to irregularly grouped, 'fragmented' sex combs (44% and 43%, respectively). By contrast, Med12^- and Med13^- clones led to slightly reduced (mean=nine teeth) but mostly aligned sex combs (Loncle, 2007).

Regarding leg organization, mutant clones for the four genes differentially affected overall size as well as formation of the joints separating the five tarsi along the proximo-distal axis. Med12^- and Med13^- clones provoked strong distal leg shortening, whereas Cdk8^- and CycC^- showed little effect. The most proximal joint, separating the first and second tarsi, was affected in a majority of T1 legs for all four genotypes (53%-94% defective or deleted). In the three more distal joints, however, the effects of Med12^- and Med13^- clones were markedly stronger (92%-100%) than Cdk8^- and CycC^- (4%-36%) (Loncle, 2007).

Taken together, these data lead to several conclusions. First, all four genes encoding subunits of the putative Cdk8 module are required for localized functions in normal leg development. Second, Cdk8 and CycC have nearly identical mutant phenotypes, indicating that they function as obligatory partners in vivo as expected for a specific Cdk-cyclin pair. Similarly, the identical mutant leg phenotypes for Med12 and Med13 reinforce the interpretation that they also function together as a pair, as previously reported for the eye and the wings (Treisman, 2001; Janody, 2003). Third, the mutant phenotypes owing to Cdk8-CycC differ from those of Med12-Med13. This result is contrary to expectations if these module components serve exclusively to regulate Cdk8 kinase activity as in S. cerevisiae. These observations thus strongly suggested the possibility that the Cdk8-CycC and Med12-Med13 pairs have independent functions in Drosophila (Loncle, 2007).

To ask whether the in vivo effects of the Cdk8-CycC pair or of its molecular partners Med12-Med13 on tarsal segmentation reflect Cdk8 module function, the epistatic relationships between the two pairs was tested. Taking advantage of the fact that Cdk8 and Med12 are on the same chromosome arm, the double mutant was generated and the distal leg phenotypes of Cdk8^- Med12^- clones were compared with those due to single mutant clones. The effects provoked by Cdk8^- Med12^- double mutant clones were indistinguishable from Med12^- alone but distinct from their Cdk8^- counterparts, both for sex comb differentiation and for overall leg structure. This indicates that Med12 is epistatic to Cdk8. Clones of Cdk8^- cells lead to a characteristic fragmented sex comb only in the presence of normal Med12 function. This observation supports the interpretation that the Cdk8-CycC pair functions together with the Med12-Med13 pair within the Cdk8 module in this developmental program. Conversely, the distal tarsal defects specific to Med12^- clones are not modified in Cdk8^- Med12^- double mutant clones. This shows that Med12 does not require Cdk8 to exert its effects there. These data thus support the existence of both linked and independent functions for the Cdk8 module components (Loncle, 2007).

To identify molecular targets of the Cdk8 module subunits, leg patterning genes were sought whose expression in L3 imaginal discs is altered in mutant clones. Tarsal segmentation is specified through a relatively well-described genetic cascade. Among a number of candidate genes examined, the expression patterns of most regional markers were unaffected in clones of mutant cells. Such markers included decapentaplegic (dpp), wingless, Dll and dachshund (dac). By contrast, regional expression of the bric-à-brac-2 (bab2) gene was markedly altered. The bab2 gene encodes a BTB-class transcription factor that is required for elaboration of the proximo-distal leg axis and is expressed in concentric rings of cells prefiguring each tarsal segment. Mutant clones for Med12 or Med13 in this region cell-autonomously downregulated bab2 expression. In contrast, bab2 expression was not affected either for small clones of Cdk8^- or CycC^- cells or for large, Minute-enhanced clones that occupied nearly the entire leg disc. These data establish that both Med12 and Med13, but not Cdk8 or CycC, are required to activate and/or maintain bab2 expression. Accordingly, bab2 is downregulated in Cdk8^- Med12^- double mutant cells as for Med12^- alone (Loncle, 2007).

To examine Cdk8 module functions in another adult tissue, clones of Cdk8^-, CycC^-, Med12^-, Med13^- or Cdk8^- Med12^- cells were generated in the developing eye. As previously reported (Treisman, 2001), large Med12^- or Med13^- clones failed to differentiate ommatidia. Further, Med12^- or Med13^- clones cell-autonomously misexpressed two early-acting eye patterning genes, dac and dpp, failing to activate these targets within the morphogenetic furrow (MF) or to repress them more posteriorly (Treisman, 2001). By contrast, adults harboring large Cdk8^- or CycC^- clones developed full-sized eyes with mostly normal differentiated ommatidia. Consistent with this, clones of Cdk8^- or CycC^- cells showed no effect on dac expression anywhere in the eye disc. As for the legs, the effects of Cdk8^- Med12^- double mutant clones were indiscernible from Med12^- alone. Misexpression of dac was still observed for Cdk8^- Med12^- clones both within and posterior to the MF. It is concluded that Med12-Med13 act independently of Cdk8-CycC in early eye differentiation (Loncle, 2007).

Although the results described indicate that Med12-Med13 and Cdk8-CycC pairs can act independently in developing legs and eyes, no evidence conclusively supported shared functions. Having observed that partial rescue of CycC^- mutants by transgenic constructs resulted in adults with reduced numbers of external sensory organs including macrochaetes and microchaetes, bristle specification/differentiation was examined for the adult notum. In preliminary experiments where mutant clones were generated in L3 larvae by heat-pulse-induced Flp recombinase, clones of Med12^-, Med13^-, Cdk8^- or CycC^- cells were all associated with localized loss of bristles. Macrochaete specification occurs in late L3, at a time when normal Cdk8 and CycC proteins are no longer detected in mutants. Therefore the effects of representatives of each Cdk8 module pair, Med12 and Cdk8, on macrochaete development was analyzed in greater detail. Minute-enhanced clones of Med12^- or Cdk8^- cells were induced in the dorsal compartment of the wing discs, coupling the ap-Gal4 driver with an UAS-Flp element. Whereas the notum normally show a stereotyped pattern of bristles, animals harboring large clones of Med12^- or Cdk8^- cells displayed extensive loss of macrochaetes. Taken together, these data support an action of Med12-Med13 in external sensory organ specification/differentiation that is shared with Cdk8-CycC (Loncle, 2007).

To identify molecular targets coregulated by the four Cdk8 module subunits, candidate genes known to be required for peripheral nervous system (PNS) development were examined. The PNS organs are generated by asymmetric divisions from an initially specified sensory organ precursor (SOP). A specific marker for SOPs and their descendant cells is the expression of the senseless (sens) gene product, a zinc-finger transcription factor that is specifically expressed in SOPs and required for their normal development. Sens protein expression was mostly absent from mosaic L3 wing discs in clones of Med12^- or Cdk8^- cells. Taken together, these results indicate a shared role of Med12-Med13 and Cdk8-CycC pairs in normal regulation of sens during SOP specification/differentiation (Loncle, 2007).

This study has investigated the functions of the four Cdk8 module subunits in Drosophila, comparing in vivo defects induced by null mutants of Cdk8, CycC, Med12 and Med13. Each gene is essential for the development of the organism but not for cell viability. The observation that fly Cdk8 and CycC proteins interact with Med12 and Med13 in vitro and/or in yeast cells reinforces the notion of a conserved Cdk8 module deduced from whole-genome analyses (Boube, 2002). The genetic data provide evidence that the four proteins composing the fly Cdk8 module can act together in vivo, as seen for the regulation of sens in sensory organ development. However, their divergent effects in regulating the target genes bab2, dac and dpp during tarsal and eye differentiation leads to the inference of a functional diversification of Cdk8-CycC and Med12-Med13 into two distinct pairs possessing diverging functions within the confines of a shared MED module. Interestingly, whereas Med12 and Med13 are specifically required for a temporally restricted regulation of dac and dpp in eye disc cells, the same patterning genes are unaffected in leg disc mutant cells. This indicates that Med12-Med13 activity depends both on the target genes and the developmental context (Loncle, 2007).

The prevailing view of Cdk8 module action, based on the functional comparison of its four components in the budding yeast, holds that this MED module serves exclusively to ensure regulated kinase activity (Myer, 1998). Interestingly, recent work has raised the possibility of a Cdk8-independent Med13 activity situated downstream of the S. cerevisiae Ras/PKA signaling pathway (Chang, 2004). Comparative analysis has revealed that Drosophila Med12-Med13 can act independently of Cdk8-CycC. One possible explanation is that another Cdk and/or cyclin may partially replace these proteins within the fly 'Cdk8' module. However, whereas purified mammalian MED complexes contain the Cdk8-related subunit Cdk11 (Conaway, 2005) and human Cdk3 can interact with CycC in cultured cells (Ren, 2004), neither Cdk11 nor Cdk3 has a counterpart in the D. melanogaster genome. Furthermore, the equivalent mutant phenotypes of Cdk8 and CycC in the current in vivo analysis support the idea that Cdk8 or CycC do not associate with another cyclin or Cdk. It is concluded that Drosophila Med12-Med13 likely function independently of a Cdk kinase activity in vivo (Loncle, 2007).

The preceding considerations lead to the deduction that Med12 and Med13 are able to associate with the core MED independently of Cdk8 or CycC. Conversely, it has been proposed that S. cerevisiae Med12 and Med13 are required for the stable association of the Cdk8-CycC pair to core MED (Myer, 1998). Accordingly, MED from human cells depleted for Med12 also exhibits diminished Cdk8 (Kim, 2006). In the present work, both the observed binding of fly Med12 and Med13 to Cdk8 and CycC in vitro and the genetic dependency of Cdk8 on Med12 in vivo reinforce this model. Importantly, although the data are consistent with a role of Med12-Med13 proteins in associating the Cdk8-CycC pair to core MED, they above all highlight that Med12-Med13 also act independently of Cdk8 or CycC (Loncle, 2007).

The biochemical nature of this Cdk-independent activity of Med12-Med13 remains to be deciphered. It seems likely that the large Med12-Med13 pair (~500 kDa in metazoans) directly contributes to the extensive structural rearrangements occurring within MED on binding to specific activators (Taatjes, 2004). Consistent with this idea, mammalian Med12 physically interacts with diverse transcriptional regulators. Such Cdk-independent regulatory activity of Med12 and Med13 may directly impact the interaction of core MED with PolII. It is speculated that kinase-independent Med12-Med13 activities may have contributed extensively during evolution to regulate and diversify cell differentiation processes (Loncle, 2007).

Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13

Wnt target gene transcription is mediated by nuclear translocation of stabilized β-catenin, which binds to TCF and recruits Pygopus, a cofactor with an unknown mechanism of action. The mediator complex is essential for the transcription of RNA polymerase II-dependent genes; it associates with an accessory subcomplex consisting of the Med12, Med13, Cdk8, and Cyclin C subunits. The Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo and skuld, are essential for the transcription of Wingless target genes. kohtalo and skuld act downstream of β-catenin stabilization both in vivo and in cell culture. They are required for transcriptional activation by the N-terminal domain of Pygopus, and their physical interaction with Pygopus depends on this domain. It is proposed that Pygopus promotes Wnt target gene transcription by recruiting the mediator complex through interactions with Med12 and Med13 (Carrera, 2008).

The mediator complex was first defined in yeast as a large multisubunit complex required for transcription of RNA polymerase II (PolII)-dependent genes. Since then, its composition and function have been shown to be conserved in Drosophila, mouse, and human cells. The mediator complex can directly bind to Pol II and recruit it to target promoters, but it also appears to function at a step subsequent to Pol II assembly into the preinitiation complex (Wang, 2005; Hu, 2006). Several mediator subunits have been shown to act as adaptors for specific transcription factors, linking them to the mediator complex and allowing them to activate transcription (Kato, 2002; Yuan, 1998; Stevens, 2002; Yang, 2004; Kim, 2004; Yang, 2006; Carrera, 2008 and references therein).

Four subunits, Med12, Med13, Cdk8, and Cyclin C (CycC), form an accessory subcomplex known as the kinase module. Genetic and microarray analyses in yeast implicate the kinase module primarily in transcriptional repression. Many of its effects have been attributed to the Cdk8 kinase, which phosphorylates the C-terminal domain of Pol II, the Cyclin H component of the TFIIH general transcription factor, and other subunits of the mediator complex, as well as specific transcription factors. The large Med12 and Med13 proteins are required for specific developmental processes in Drosophila, zebrafish, and Caenorhabditis elegans, but their biochemical functions are not understood (Carrera, 2008 and references therein).

Secreted proteins of the Wnt family play important roles in both development and oncogenesis. Transcription of Wnt target genes is mediated by nuclear translocation of stabilized Armadillo (Arm)/β-catenin and its binding to the HMG box transcription factor TCF. The adaptor protein Legless (Lgl)/Bcl-9 links Armβ-catenin to Pygopus (Pygo); the N-terminal homology domain (NHD) of Pygo is essential for Wnt-regulated transcriptional activation and is thought to interact with unknown general transcriptional regulators. This study shows that the Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo (kto) and skuld (skd) (Treisman, 2001), are essential for the transcription of Wingless (Wg) target genes in vivo and a Wg-responsive reporter in cultured cells. skd and kto act downstream of Arm stabilization and are required for the function of the NHD of Pygo when fused to an exogenous DNA-binding domain. Skd and Kto interact with Pygo in vivo through the NHD. It is suggested that this interaction recruits the mediator complex to allow for the transcription of Wg target genes (Carrera, 2008).

Two domains of Arm/α-catenin are important for the activation of Wnt target genes: (1) Arm repeats 1-4, which act by binding Lgs and thus recruiting Pygo, and (2) a C-terminal transcriptional activation domain. The C-terminal domain has been shown to bind to the histone acetyltransferases p300 and CBP, Hyrax/Parafibromin, which recruits histone modification complexes, and directly to the Med12 mediator complex subunit (Kim, 2006). However, this domain is insufficient for target gene activation in vivo, which requires Lgs, Pygo, and an amino acid in Arm that is critical for Lgs binding. In addition, although the C-terminal domain is a strong activator in cell culture, it is not sufficient to replace the function of Arm in vivo when fused to dTCF, whereas the activation domain of Pygo is. It has been proposed that Pygo interacts with unidentified general transcriptional regulators through its NHD (Kramps, 2002). The current results suggest that the Pygo NHD recruits the mediator complex through the Kto/Med12 and Skd/Med13 subunits and that these subunits are essential for its activation function (Carrera, 2008).

An alternative view of the role of Pygo is that it acts as a nuclear anchor for Lgs and Arm. This model has been further refined by recent data showing that Pygo is constitutively localized to Wg target genes in a manner dependent on its NHD and on TCF, and it might function there to capture Arm. However, the finding that PygoDeltaPHD-GAL4 is sufficient to activate UAS-GFP expression in all cells in vivo strongly supports an additional activation function for Pygo. It is suggested that this function reflects its ability to recruit the mediator complex. Interestingly, the C. elegans Med12 and Med13 homologues have been implicated in the transcriptional repression of Wnt target genes although these effects have not been shown to be direct (Yoda, 2005; Zhang, 2000). Their dispensability for Wnt target gene activation may reflect the absence of pygo homologues in the worm genome (Carrera, 2008).

The kinase module of the mediator complex is commonly thought to have a repressive function; it has been shown to sterically hinder recruitment of Pol II (Elmlund, 2006), and Ras signaling promotes transcriptional elongation by inducing loss of this module from the mediator complex bound to C/EBP-regulated promoters (Mo, 2004). However, recent results suggest that the kinase module can play a role in transcriptional activation as well as repression (Larschan, 2005; Donner, 2007). An exclusively repressive function would be difficult to reconcile with the observation that the genome-wide occupancy profiles of Cdk8 and Med13 characterized by ChIP match that of the core mediator complex. The current results support an essential and direct function for the Med12 and Med13 subunits in the activation of Wg target genes. The transcriptional and phenotypic profiles of mutants in the four subunits of the yeast kinase module are very similar. However, Drosophila cdk8 and cycC are required for only a subset of the functions of skd and kto (Loncle, 2007) that does not include Wg target gene activation. Therefore, Med12 and Med13 may have gained additional functions during the evolution of higher eukaryotes. The identical defects of the two mutants may reflect the requirement for Skd to stabilize the Kto protein. Similarly, Med24 stabilizes Med16 and Med23 and promotes their incorporation into the mediator complex (Carrera, 2008 and references therein).

Several mediator complex subunits act as adaptors that link specific transcription factors to the mediator complex. For example, Med1 interacts with nuclear receptors; Med23 interacts with phosphorylated Elk-1, the adenovirus E1A protein, and Heat shock factor; Med16 interacts with differentiation-inducing factor; and Med15 interacts with Smad2/3 and Sterol regulatory element-binding protein. The current results show that, despite their location in a module that is not part of the core mediator complex, Med12 and Med13 act as adaptors for Pygo. These subunits also are likely to act as adaptors for additional transcription factors because mutations in Drosophila and other organisms have other phenotypes that cannot be explained by loss of Wg signaling. Indeed, Med12 has been shown to interact with both Sox9 and Gli3. The yeast Med13 homologue is a target for Ras-regulated PKA phosphorylation, suggesting the interesting possibility that Wg or other signals might directly regulate the activity of Med12 or Med13. Finally, because skd and kto are not essential for normal cell proliferation or survival, they may provide targets for the treatment of Wnt-driven cancers (Carrera, 2008).

Requirements for mediator complex subunits distinguish three classes of notch target genes at the Drosophila wing margin

Spatial and temporal gene regulation relies on a combinatorial code of sequence-specific transcription factors that must be integrated by the general transcriptional machinery. A key link between the two is the mediator complex, which consists of a core complex that reversibly associates with the accessory kinase module. Genes activated by Notch signaling at the dorsal-ventral boundary of the Drosophila wing disc fall into three classes that are affected differently by the loss of kinase module subunits. One class requires all four kinase module subunits for activation, while the others require only Med12 and Med13, either for activation or for repression. These distinctions do not result from different requirements for the Notch coactivator Mastermind or the corepressors Hairless and Groucho. It is proposed that interactions with the kinase module through distinct cofactors allow the DNA-binding protein Suppressor of Hairless to carry out both its activator and repressor functions (Janody, 2011).

Intercellular signaling pathways drive many processes during development. Their activation results in changes in transcription factor activity that lead to the activation or repression of specific target genes. An important goal is to understand the transcriptional regulatory codes that allow the combinations of proteins bound to enhancer elements to direct precise patterns of gene expression. One well-characterized developmental paradigm is the specification of the Drosophila wing margin by Notch signaling. The Notch receptor is specifically activated at the dorsal-ventral boundary of the larval wing imaginal disc, due to the restricted expression of its ligands Delta and Serrate and of the glycosyltransferase Fringe. Notch activation results in expression of the target genes Enhancer of split m8 (E(spl)m8), cut, wingless (wg), and vestigial (vg), the last through a specific enhancer element known as the boundary enhancer (vgBE). Wg signaling then leads to the differentiation of characteristic sensory bristles adjacent to the margin of the adult wing (Janody, 2011).

Upon ligand binding, Notch is cleaved by the γ-secretase complex, and its intracellular domain (Nintra) enters the nucleus, where it interacts with the DNA-binding protein Suppressor of Hairless (Su(H)). In the absence of Notch activation, Su(H) represses target gene expression through interactions with the corepressor Hairless (H), which binds to Groucho (Gro) and C-terminal binding protein (CtBP). Nintra displaces these corepressors from Su(H) and recruits coactivators such as Mastermind (Mam). It has been proposed that only a subset of Notch target genes require Su(H) to recruit coactivators, while others require Notch signaling only to relieve Su(H)-mediated repression, allowing transcription to be activated by other factors. However, the mechanisms by which Su(H) directs both activation and repression are not fully understood (Janody, 2011).

The mediator complex is thought to promote transcriptional activation by recruiting RNA polymerase II (Pol II), the general transcriptional machinery, and the histone acetyltransferase p300 to promoters, and by stimulating transcriptional elongation by Pol II molecules paused downstream of the promoter. The 'head' and 'middle' modules of the core complex bind to Pol II and general transcription factors, while the 'tail' module consists largely of adaptor subunits that bind to sequence-specific transcription factors. This core complex reversibly associates with a fourth 'kinase' module that consists of the four subunits Med12, Med13, Cdk8, and Cyclin C (CycC). Several studies have implicated the kinase module in transcriptional repression, which can be mediated by phosphorylation of Pol II and other factors by Cdk8, by histone methyltransferase recruitment, and by occlusion of the Pol II binding site. However, this module also appears to function in activation in some contexts; for example, it promotes Wnt target gene expression during Drosophila and mouse development, in mammalian cells, and in colon cancer. Although all four subunits have very similar mutant phenotypes in yeast, loss of Med12 or Med13 has more severe effects on Drosophila development than loss of Cdk8 or CycC, suggesting that Med12 and Med13 have evolved additional functions in higher eukaryotes (Janody, 2011).

This study shows that Notch target genes at the wing margin can be divided into three classes based on their requirements for kinase module subunits. An E(spl)m8 reporter requires all four subunits for its activation, cut requires only Med12 and Med13 (known as Kohtalo [Kto] and Skuld [Skd], respectively, in Drosophila) for its activation, and wg and the vgBE enhancer require Med12 and Med13 for their repression in cells close to the wing margin. Because Med12 and Med13 coimmunoprecipitate with Su(H), regulate an artificial reporter driven by Su(H) binding sites, and can be replaced by a VP16 activation domain or a WRPW repression signal fused to Su(H), it is proposed that the kinase module directly regulates Notch target genes. All four Notch target genes fail to be expressed in the absence of Mam and are similarly affected by the loss of Hairless or Gro, suggesting that other more specific cofactors might recruit kinase module subunits to these genes (Janody, 2011).

The kinase module of the mediator complex is conserved throughout eukaryotes, yet its functions in transcription remain poorly understood. In yeast, loss of any of the four subunits has a very similar effect. In Drosophila, however, loss of Med12 or Med13 has more dramatic effects than loss of Cdk8 or CycC. The kinase module was originally thought to be primarily important for transcriptional repression, mediated by the kinase activity of Cdk8. However, Med12 and Med13 appear to directly activate genes regulated by Wnt signaling in Drosophila and mammalian systems, and also play a positive role in gene activation by the Gli3 and Nanog transcription factors. The data presented in this study confirm that Med12 and Med13 have functions distinct from Cdk8 and CycC. In addition, evidence is provided that all four kinase module subunits contribute to the activation of E(spl)m8 (Janody, 2011).

The human Mastermind homologue MAM has been shown to recruit Cdk8 and CycC to promoters of Notch target genes, where Cdk8 phosphorylates the intracellular domain of Notch, leading to its ubiquitination by the Fbw7 ligase and degradation (Fryer, 2004). This mechanism would be expected to reduce Notch target gene expression, consistent with the increase in E(spl)mβ expression seen in clones lacking the Drosophila Fbw7 homologue Archipelago (Nicholson, 2011); thus it cannot explain the positive effects of Cdk8 and CycC on E(spl)m8. A function for Cdk8 and CycC in Notch-mediated activation would be analogous to recent findings showing that Cdk8 phosphorylation of Smad transcription factors and of histone H3 promotes activation. Cdk8 phosphorylation of RNA polymerase II (Pol II) is also important for transcriptional elongation (Janody, 2011).

Of interest, the current data also suggest that Med12 and Med13 are involved in the repression of wg and the vgBE enhancer in the absence of Notch signaling. The kinase module has been proposed to inhibit transcription through steric hindrance of Pol II binding, independently of Cdk8 kinase activity. Removal of this module on the C/EBP promoter is thought to convert the mediator complex to its active form. In contrast, this study find that wg and vgBE require Med12 and Med13 for their repression but not their activation, while cut and E(spl)m8 require Med12 and Med13 only for their activation, arguing that the two functions occur on different promoters. It cannot be ruled out that Med12 and Med13 have only indirect effects on some of the genes examined; however, their physical association with Su(H) and the requirement for Su(H) binding sites for misexpression of an artificial reporter in skd and kto mutant clones are consistent with a direct effect of Med12 and Med13 on the Su(H) complex (Janody, 2011).

Med12 and Med13 are found associated with both active and inactive promoters in genome-wide chromatin immunoprecipitation studies, suggesting that they can have different effects on transcription when bound to distinct interaction partners. Although both are very large proteins, they contain no domains predicted to have enzymatic activity, and may instead act as scaffolds for the assembly of transcriptional complexes (Janody, 2011).

It has been proposed that Notch target genes could be categorized into two classes: permissive genes, for which the primary function of Notch is to relieve repression by the Su(H) complex, and instructive genes, for which Notch plays an essential role in activation by recruiting specific coactivators. These differences presumably depend on the combinatorial code of transcription factors that regulate each promoter. This study shows that vgBE, an enhancer previously placed in the permissive category, as well as wg, require Med12 and Med13 for their repression but not their activation. During eye development, the proneural gene atonal is likewise regulated permissively by Notch, and ectopically expressed in skd or kto mutant clones. Unexpectedly, this study found that Gro, previously thought to be a cofactor through which Hairless mediates repression, is not required for the repression of vgBE or wg. Hairless may repress target genes at the wing margin through CtBP, its other binding partner. Alternatively, Gro may affect the expression of other upstream regulators of wing margin fate, masking its repressive effect on the genes that were examined (Janody, 2011).

It was also show in this study that instructive Notch target genes can be further subdivided into two classes based on their requirement for kinase module subunits; E(spl)m8 requires all four subunits, while cut requires Med12 and Med13, but not Cdk8 and CycC. Cdk8 and CycC may simply increase the ability of the mediator complex to recruit Pol II or promote transcriptional initiation; this model would suggest that E(spl)m8 has a higher activation threshold than cut. Alternatively, Cdk8 and CycC might enhance the function of a transcription factor that is specifically required for the expression of E(spl)m8 but not cut. Good candidates for such factors would be the proneural proteins Achaete or Scute or their partner Daughterless (Janody, 2011).

The mechanism by which the kinase module is recruited to promote the activation of instructive target genes is not yet clear. Although Mam proteins are well-characterized coactivators for N_intra, this study found that Mam is necessary for the activation of both instructive and permissive genes. It may thus have a general function in transcriptional activation, such as recruiting histone acetyltransferases or stabilizing the Notch-Su(H) complex. A coactivator that recruits Med12 and Med13 specifically to instructive target genes to promote activation may remain to be identified. The current results, like recent reports demonstrating that the arrangement of Su(H) binding sites can affect the interactions between Notch and its coactivators, highlight the complexity in the mechanisms through which promoter elements respond to Notch signaling (Janody, 2011).

Drosophila Cdk8, a kinase partner of cyclin C that interacts with the large subunit of RNA polymerase II

A number of cyclins have been described, most of which act together with their catalytic partners, the cyclin-dependent kinases (Cdks), to regulate events in the eukaryotic cell cycle. Cyclin C was originally identified by a genetic screen for human and Drosophila cDNAs that complement a triple knock-out of the CLN genes in Saccharomyces cerevisiae. Unlike other cyclins identified in this complementation screen, there has been no evidence that cyclin C has a cell-cycle role in the cognate organism. This study reports that cyclin C is a nuclear protein present in a multiprotein complex. It interacts both in vitro and in vivo with Cdk8, a novel protein-kinase of the Cdk family, structurally related to the yeast Srb10 kinase. Cdk8 can interact in vivo with the large subunit of RNA polymerase II and a kinase activity that phosphorylates the RNA polymerase II large subunit is present in Cdk8 immunoprecipitates. Based on these observations and sequence similarity to the kinase/cyclin pair Srb10/Srb11 in S. cerevisiae, it is suggested that cyclin C and Cdk8 control RNA polymerase II function (Leclerc, 1996).

Anti-cyclin C antibodies identified cyclin C as a nuclear protein whose level does not oscillate during the blastoderm cell cycles in Drosophila embryos. Because analysis of the same embryos with cyclin A and cyclin B antisera revealed mitotic destruction of these cyclins, it appears that cyclin C is not subject to the degradation that drives oscillation of these cyclins. Absence of mitotic destruction was further supported by cyclin C immunolocalization in the embryo: antigen levels were constant throughout interphase nuclei, and at mitosis there was striking localization of the antigen to mitotic chromosomes. The protein, which migrates in SDS-PAGE with an apparent molecular mass of 28 kDa, sediments on native sucrose gradients with an estimated mass of ~500 kDa, which indicates that cyclin C is part of a large multiprotein complex. This unusually large size allowed the co-sedimentation of members of the Cdk family and other cell cycle regulators with cyclin C fractions to be tested. Neither DmCdc2, DmCdc2c (alias DmCdk2), cyclin A, nor cyclin B co-sedimented with cyclin C. They were present in complexes in the 100- to 200-kDa range as expected from sedimentation results. Consequently, the co-sedimentation of cyclin C and a unique 51-kDa band recognized by anti-human Cdk8 antibodies strongly suggested in vivo interaction between these two molecules; this was confirmed later by co-immunoprecipitation of DmCdk8 and cyclin C from nuclear extracts. It also falls in line with the recent finding that human Cdk8 interacts specifically with cyclin C in HeLa cell lysates (Leclerc, 1996).

Based on sequence data for a human Cdk8 and the yeast Ume5/SrblO protein, Drosophila sequences were amplified by PCR, and a cDNA clone for the DmCDK8 gene was isolated and sequenced. The human and Drosophila Cdk8 aminoacid sequences show numerous identities, and comparison to SrblO and other Cdks leads to a few remarks. The classical PSTAIRE sequence that is involved in Cdk-cyclin interactions is replaced in both Cdk8 and SrblO by a S(M/Q)SACRE sequence, whose conservation could reflect the presence of a contact region for related cyclins like Srbl 1 and cyclin C. The 'T-loop' region of the Cdks is regulatory: in the inactive structure it virtually blocks the active site, while the active site is unmasked in the cyclin-bound structure. Full activation of characterized Cdks requires phosphorylation of the T-loop. Although the structure of the phosphorylated enzyme has not yet been solved, based on an analogy to the structure of the cyclic AMP-induced kinase, it has been proposed that phosphorylation introduces new interactions that stabilize the active conformation of the T-loop. Cdk8 of human and Drosophila appear to lack a requirement for this activating phosphorylation because the T-loops of these kinases lack candidate residues for phosphorylation (S, T, or Y). Although SrblO has a T within its T-loop, it is not clear whether it is analogous to the phosphorylated residue in other Cdks. The ambiguity arises because both Cdk8's and SrblO have a three-aminoacid insertion within the T-loop, and the level of homology is not sufficient to define the correct alignment. Most likely the change in the T-loop occurred at the site that is usually phosphorylated because structural comparison indicates that adjacent residues are involved in highly conserved structure. It is suggested that the Cdk8 kinases as well as the SrblO kinase do not require activating phosphorylation and that the Asp residue that has been introduced roughly at the position normally occupied by Thr provides a negative charge to substitute for phosphorylation (Leclerc, 1996).

Two results showed that the cloned Drosophila Cdk8 is specifically recognized by the antibody to the human Cdk8: in vitro-translated DmCdk8 protein was recognized by anti-human Cdk8 antibodies and comigrated on SDS-PAGE with a 51-kDa band seen in extracts. Western blots using this antibody showed that DmCdk8 co-sediments with cyclin C in sucrose gradients and immunoprecipitations showed that cyclin C co-precipitates with DmCdk8, suggesting that these proteins might work in association to produce an active kinase complex. Indeed, a kinase activity was found in anti-Cdk8 immunoprecipitate that phosphorylates a 240-kDa band that co-migrates with form IIo of RNA Pol II. It was confirmed that this band corresponded to form IIo of RNA Pol II by four independent lines of evidence. (1) RNA Pol II is present in the immunoprecipitates. (2) Depleting the extracts before immunoprecipitation with anti-Cdk8 antibodies suppresses the appearance of the 240-kDa phosphorylated band. (3) The 240-kDa phosphoprotein co-migrates exactly with form IIo of RNA Pol II run on the same gel. (4) Cdk8 immunocomplexes are capable of phosphorylating a CTD peptide in vitro (Leclerc, 1996).

Attempts to precipitate the cyclin C/DmCdk8 complex with two different antibodies to RNA polymerase failed. In the case of the ARNA3 antibodies, the failure can be simply explained because under the experimental conditions used no precipitation of the large subunit of RNA Pol II was seen. Anti-CTD antibodies efficiently immunoprecipitated Pol II in the extracts but no trace of Cdk8 or cyclin C were detected in the immunoprecipitate. Failure to co-immunoprecipitate both Cdk8 and cyclin C using anti-CTD antibodies might be due to a variety of reasons, but it might reflect a competition effect between antibody molecules and the proteins interacting with the CTD epitopes. A similar effect was previously suggested to explain the separation of the RNA Pol II core enzyme and a group of associated proteins called 'the mediator' (and among them, SrblO and Srbll) during affinity purification experiments using anti-CTD antibodies (Leclerc, 1996).

These experiments do not resolve whether phosphorylation of Pol II is due to the activity of the Cdk8 kinase itself. Other kinase activities might be present in the immune complex. For this reason, the presence was tested of a kinase previously implicated in phosphorylation of the CTD. Transcription factor TFIIH, which is involved in the phosphorylation of the CTD, contains Cdk7 and cyclin H. This pair of proteins is responsible for the in vitro CTD-kinase activity of the TFIIH complex. No trace of Drosophila Cdk7 protein was detected in the anti-Cdk8 immune complex using affinity- purified anti-DmCdk7 antibodies. This suggests either that the experimental conditions do not allow the stabilization of a complex comprising Cdk7 and Cdk8, or that the two kinases associate separately with the RNA Pol II enzyme. It indicates, furthermore, that the kinase activity present in the anti-Cdk8 immune precipitate is not due to the Cdk7 kinase (Leclerc, 1996).

In conclusion, this study has identified Cdk8, a kinase partner of cyclin C, and has demonstrated that it can interact in vivo with the large subunit of RNA Pol II. Furthermore, DmCdk8 is associated either directly or indirectly with a kinase activity that can phosphorylate the large subunit of RNA Pol II in vitro. The structural identity between Cdk8/cyclin C in Drosophila and SRB10/SRB11 in yeast, as well as the in vivo interaction between RNA Pol II and Cdk8 lead to the suggestion of a possible functional homology between the two kinase/cyclin pairs. Recent results assign a role for SRB10/SRB11 in transcriptional regulation in vivo as well as CTD phosphorylation in vitro. Drosophila genetics as well as additional biochemical studies will help to define the precise function of Cdk8/cyclin C. The possible involvement of cyclin C and Cdk8 in transcription suggests that the basis of the original isolation of cyclin C as a gene capable of complementing deficiencies in Gl cyclins in yeast should be re-evaluated. Although it is possible that cyclin C can play a cell cycle role in a foreign context, it is also possible that cyclin C function bypassed the Gl block by disturbing transcriptional controls in yeast (Leclerc, 1996).

Drosophila mediator complex is used by Heat shock factor

To decipher the mechanistic roles of Mediator proteins in regulating developmental specific gene expression and compare them to those of TATA-binding protein (TBP)-associated factors (TAFs), a multiprotein complex containing Drosophila Mediator (dMediator) homologs was isolated and analyzed. dMediator interacts with several sequence-specific transcription factors and basal transcription machinery and is critical for activated transcription in response to diverse transcriptional activators. The requirement for dMediator does not depend on a specific core promoter organization. By contrast, TAFs are preferentially utilized by promoters having a specific core element organization. Therefore, Mediator proteins are suggested to act as a pivotal coactivator that integrates promoter-specific activation signals to the basal transcription machinery (Park, 2001).

Previous studies in yeast and human cells have suggested that transcriptional activator proteins interact with Mediator complexes. The requirement of dMediator for the activated transcription in response to Gal4-VP16 indicates that dMediator may also serve as a binding target of transcriptional activators. Because several coactivators, such as TAFs and the GCN5 histone acetyltransferase (HAT) complex, have been suggested to interact directly with transcriptional activators, the relative binding affinities of these coactivator complexes with the VP16 protein were examined. After incubation of nuclear extracts with an excess of GST fusion protein beads containing either wild-type or mutant (Delta456FP442) VP16 activation domain, the supernatants were analyzed by immunoblotting with Abs against the components of the coactivator complexes. Almost all of the dMediator proteins in the nuclear extract (TRAP80, MED6, and Trfp) were removed by incubating with GST-VP16 but not with GST-VP16^{Delta456FP442}. However, the amounts of dGCN5, dTAF_II40, dTAF_II250, and dTBP in the extract were not reduced at all by the incubation. When the proteins bound to the beads were analyzed, a large amount of dMediator was retained only in the GST beads containing the functional VP16 activation domain. The TFIID and dGCN5 HAT complexes did bind to the wild-type VP16 beads, but the amounts were less than 2% of the total amounts present in the extract. These data indicate that, among known transcriptional coactivator complexes, Mediator is most strongly bound to and most readily recruited to the activation domain (Park, 2001).

In addition to the model VP16 activator derived from herpesvirus, dMediator interacts with Drosophila transcriptional activators Dorsal and heat shock factor (dHSF). When dMediator complex was incubated with FLAG-Dorsal or GST-dHSF fusion protein beads, more than 20% of the dMediator input was retained specifically on the beads even after extensive washing. To extend this study to other sequence-specific transcription factors important for Drosophila development, dMediator was immobilized on protein G-agarose beads through anti-dSOH1 Ab and the binding of diverse ³⁵S-labeled Drosophila transcription factors was examined. Bicoid, Krüppel, and Fushi-tarazu are retained specifically on the dMediator beads; Twist and Hunchback are not. Therefore, dMediator functions as a binding target for many, but not all, developmental specific transcription factors (Park, 2001).

To evaluate the requirement of dMediator for activated transcription in response to the Drosophila activator proteins that interact with dMediator, the ability of dMediator-deficient nuclear extracts to support transcriptional activation by the Dorsal and Gal4-dHSF proteins was examined. The addition of Dorsal or Gal4-dHSF to mock-depleted extract causes 20- and 25-fold increases, respectively, in transcription levels from the adenovirus early region 4 (E4) promoter linked to the appropriate DNA binding site. However, the level of transcriptional activation is reduced significantly (five- and three-fold activations, respectively) in nuclear extract that has been depleted by anti-dSOH1 Ab. Therefore, dMediator is absolutely required for transcriptional activation by all the activators tested. Addition of purified dMediator back to depleted extracts partially recovers activation by Dorsal and Gal4-dHSF in much the same way as it does in the case of Gal4-VP16. dMediator is not required for transcriptional repression by the sequence-specific transcription factor Even-skipped (Park, 2001).

dMediator is generally required for transcriptional activation from both TATA-containing and TATA-less promoters through direct communication with transcriptional activators. The function of dMediator seems to be exclusively related to sequence-specific transcription factors placed at upstream enhancer elements. However, the requirement of TAFs, or at least dTAF_II250, in activated transcription appears to be redundant in the in vitro transcription system used and affected by such factors as the core promoter organization or nucleosomal structure of transcriptional templates. Several TAF components in the TFIID complex indeed have biochemical activities and structural motifs adequate for the recognition of specialized settings of transcription templates. For example, certain TAFs recognize the Inr and DPE sequences located in many Drosophila core promoters and increase the stability of TFIID-promoter interactions. In addition, TFIID contains dTAF_II250, which has a HAT catalytic activity and also possesses a histone octamer-like module comprising the histone H2B-, H3-, and H4-like TAFs. Although not experimentally demonstrated, these TAFs may have some roles in the transcriptional regulation of nucleosomal templates (Park, 2001).

The sequence-specific transcription factors which interact physically with dMediator include VP16, Dorsal, dHSF, Bicoid, Krüppel, and Fushi-tarazu. These factors contain different types of activation domains (acidic and glutamine-rich domains). Most of these transcription factors have been shown to activate transcription either constitutively or inducibly. It is noteworthy that dHSF interacts with and requires dMediator for transcriptional activation because previous reports have shown that transcriptional activation by HSF in yeast does not require the function of the Mediator protein Srb4. However, the recent finding that activation by HSF depends on another Mediator protein, Rgr1 (Trap170), suggests that some function of Mediator is required for HSF-mediated transcriptional activation in yeast, as well. Since Rgr1, but not Srb4, is conserved between yeast and Drosophila, transcriptional activation by HSF might utilize the conserved Rgr1 components of the Mediator complexes (Park, 2001).

Although some human Mediator complexes appear to have a negative effect on activated transcription, dMediator does not exhibit such an activity in an in vitro transcription system reconstituted with Drosophila transcription factors. In addition, Even-skipped, a well-known Drosophila transcriptional repressor, does not interact with, or depend for its transcriptional repression on dMediator. Previous reports have shown that the repression domain of Even-skipped directly targets TBP. It has also been confirmed that the TFIID complex in the nuclear extract specifically interacts with Even-skipped under the same conditions in which Even-skipped fails to interact with dMediator. Although Krüppel has a well-characterized repressor function in Drosophila development, it can also act as a transcriptional activator under certain conditions. Therefore, it is more plausible that the dMediator-Krüppel interaction observed is a part of the mechanism for transcriptional activation rather than transcriptional repression. Taken together with the fact that dMediator is dispensable for basal transcription, the lack of defect of the dMediator-depleted nuclear extracts on transcriptional repression by Even-skipped protein suggests that dMediator is required mainly for the mediation of transcriptional activation signals to the basal transcription machinery. Very recently, developmental roles of certain dMediator proteins found in the Drosophila genome database have begun to be also identified in genetic studies. Genetic interactions between dMediator proteins and a homeotic regulator Sex combs reduced implicate dMediator proteins as a transcriptional activator-specific target critical for Drosophila development (Park, 2001).

Like yeast Mediator, dMediator bind with the CTD repeats of Drosophila Pol II. This implies that though dMediator was purified separately from Pol II, these two complexes indeed interact with each other and act together during transcriptional initiation. Besides the physical interaction with Pol II, dMediator also has some binding affinity for TBP, TFIIB, TFIIE, TFIIF, and TFIIS. Such interactions may be involved in the regulation of Pol II preinitiation complex assembly. Related with this idea, it has been reported that in yeast, recruitment of general transcription factors such as TBP, TFIIB, and TFIIH to active promoters requires the function of Mediator. Also, TFIIE interacts with the Mediator protein Gal11. Further analyses will be required to clarify whether these interactions, observed both in yeast and Drosophila, participate in the control of the stepwise preinitiation complex assembly in the course of transcription activation or simply reflect the affinities between the components of preassembled Pol II holoenzymeG (Park, 2001).

dMediator contains the protein kinase component Cdk8, which can phosphorylate serine residues in the CTD. This catalytic kinase subunit seems responsible, at least in part, for the Pol II phosphorylation by dMediator. In particular, dMediator and TFIIH synergistically phosphorylate the serine 5 residue of the carboxy-terminal Pol II repeats, suggesting the presence of a functional interaction between these complexes. Given that Pol II phosphorylation at serine 5 by TFIIH has been correlated with transcriptional activation processes, the synergy in the serine 5 phosphorylation by TFIIH and dMediator may be intimately linked with the regulatory effects that the Mediator complex exerts on Pol II transcription (Park, 2001).

CDK8 is a colorectal cancer oncogene that regulates beta-catenin activity

Aberrant activation of the canonical WNT/β-catenin pathway occurs in almost all colorectal cancers and contributes to their growth, invasion and survival. Although dysregulated β-catenin activity drives colon tumorigenesis, further genetic perturbations are required to elaborate full malignant transformation. To identify genes that both modulate β-catenin activity and are essential for colon cancer cell proliferation, two loss-of-function screens were conducted in human colon cancer cells and genes identified in these screens were compared with an analysis of copy number alterations in colon cancer specimens. One of these genes, CDK8, which encodes a member of the mediator complex, is located at 13q12.13, a region of recurrent copy number gain in a substantial fraction of colon cancers. This study shows that the suppression of CDK8 expression inhibits proliferation in colon cancer cells characterized by high levels of CDK8 and β-catenin hyperactivity. CDK8 kinase activity was necessary for β-catenin-driven transformation and for expression of several β-catenin transcriptional targets. Together these observations suggest that therapeutic interventions targeting CDK8 may confer a clinical benefit in β-catenin-driven malignancies (Firestein, 2008).

To determine whether CDK8 induces cell transformation, wild-type CDK8 or a previously reported kinase-inactive substitution mutant (D173A; CDK8-KD) was overexpressed in immortal murine fibroblasts (NIH 3T3 cells). CDK8 expression induced focus formation, anchorage-independent colony growth and tumour formation in immunodeficient animals, whereas the CDK8-KD mutant failed to transform the cells. These observations confirm that CDK8 is a bona fide oncogene, the kinase activity of which is necessary for oncogenic activity (Firestein, 2008).

To dissect the relationship between CDK8 and β-catenin activity, endogenous β-catenin activity was measured in the 12 colon cancer cell lines. The RKO, COLO-741, HCA-7 and FHC cell lines do not harbour known APC or β-catenin mutations and, as predicted, had low amounts of β-catenin activity. Of these four cell lines, suppression of CDK8 induced a substantial proliferation effect only in COLO-741. Similarly, of the 12 cell lines tested, the six cell lines with the highest CDK8 elevation showed a greater dependence on β-catenin for proliferation (Firestein, 2008).

CDK8 is a cyclin-dependent kinase member of the mediator complex, which couples transcriptional regulators to the basal transcriptional machinery. To explore the role of CDK8 in modulating β-catenin transcriptional activity, it was confirmed that suppressing CDK8 with two independent, CDK8-specific small hairpin RNAs (shCDK8-1 and shCDK8-2) in another cell line, SW480, also reduced β-catenin-dependent transcriptional activity. CDK8 kinase activity depends on the co-factor cyclin C, and it was found that cyclin C knockdown preferentially affected colon cancer cell lines with chromosome 13q gain. To test whether CDK8 kinase activity is required to regulate β-catenin activity, wild-type CDK8 or dominantly interfering CDK8-KD was expressed in DLD-1^Rep cells carrying a shRNA targeting the 3'-untranslated region, shCDK8-1, and it was found that only wild-type CDK8 partially rescued the effects of suppressing endogenous CDK8. These observations demonstrate that the kinase activity of CDK8 is necessary for both CDK8-induced transformation and β-catenin driven transcription (Firestein, 2008).

The TCF-β-catenin complex regulates expression of several genes implicated in colon cancer, including MYC, AXIN2 and LEF1. Suppression of CDK8 in DLD-1 and COLO-205 cells reduced expression of each of these genes. In contrast, no changes were observed in the expression of Notch or HES-1, previously reported targets of CDK8. Thus, CDK8 modulates a subset of β-catenin-driven genes previously implicated in cancer (Firestein, 2008).

Chromatin immunoprecipitation (ChIP) was performed near two verified β-catenin/TCF binding elements (TBE) in the MYC promoter, as an example of a β-catenin regulated gene, to test whether CDK8 directly modulates MYC expression at the promoter level. It was found that CDK8 associated with the MYC promoter. It was therefore asked if loss of CDK8 binding at the MYC promoter affects the ability of β-catenin to bind at the proximal and distal TBEs. Suppression of CDK8 expression reduced the amount of β-catenin bound to the proximal element in the MYC promoter but had little effect on the amount associated with the distal element. These observations implicate CDK8 and the mediator complex as a direct regulator of β-catenin-driven transcription (Firestein, 2008).

To test whether CDK8 activity is also required for β-catenin-driven transformation, the dominantly interfering CDK8-KD mutant was expressed in transformed NIH 3T3 cells expressing a constitutively active β-catenin mutant. Disruption of CDK8 activity inhibited β-catenin-driven transformation, whereas a dominantly interfering TCF construct, previously shown to inhibit β-catenin-induced cellular transformation, only partially abrogated CDK8-mediated transformation. These observations suggest that although CDK8 is required for β-catenin-mediated transformation, the full capacity of CDK8 to transform cells may extend beyond its ability to activate β-catenin (Firestein, 2008).

This study has used an integrated approach to identify CDK8 as an oncogene in a substantial fraction of colorectal cancers and demonstrate that the kinase activity of CDK8 is essential for it to be able to regulate β-catenin-dependent transcription and transformation. These observations indicate that CDK8 acts in part by co-activating β-catenin-driven transcription in colon cancers characterized by both high CDK8 expression and β-catenin activity. Accordingly, therapeutic interventions that target the CDK8 kinase activity in such cancers may be of clinical value (Firestein, 2008).

Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-beta pathways

TGF-beta and BMP receptor kinases activate Smad transcription factors by C-terminal phosphorylation. This study identified a subsequent agonist-induced phosphorylation that plays a central dual role in Smad transcriptional activation and turnover. As receptor-activated Smads form transcriptional complexes, they are phosphorylated at an interdomain linker region by CDK8 and CDK9, which are components of transcriptional mediator and elongation complexes. These phosphorylations promote Smad transcriptional action, which in the case of Smad1 is mediated by the recruitment of YAP (Drosophila homolog: Yorkie) to the phosphorylated linker sites. An effector of the highly conserved Hippo organ size control pathway, YAP supports Smad1-dependent transcription and is required for BMP suppression of neural differentiation of mouse embryonic stem cells. The phosphorylated linker is ultimately recognized by specific ubiquitin ligases, leading to proteasome-mediated turnover of activated Smad proteins. Thus, nuclear CDK8/9 drive a cycle of Smad utilization and disposal that is an integral part of canonical BMP and TGF-beta pathways (Alarcon, 2009).

The present findings reveal a remarkable integration of Smad regulatory functions by agonist-induced, CDK8/9-mediated phosphorylation of the linker region and highlight this event as an integral feature of the transcriptional action and turnover of receptor-activated Smad proteins. Agonist-induced linker phosphorylation of R-Smads is a general feature of BMP and TGF-β pathways, occurring in all the responsive cell types examined, shortly after Smad tail phosphorylation. The evidence identifies CDK9 as the kinases involved and does not support a major role for MAPKs or cell-cycle-regulatory CDKs in this process. CDK8 and cyclinC are components of the Mediator complex that couples enhancer-binding transcriptional activators to RNAP II for transcription initiation. CDK9 and cyclinT1 constitute the P-TEFb complex, which promotes transcriptional elongation. CDK8 and CDK9 phosphorylate overlapping serine clusters in the C-terminal domain of RNAP II, a region which integrates regulatory inputs by binding proteins involved in mRNA biogenesis. Thus, CDK8 and CDK9 may provide coordinated regulation of Smad transcriptional activators and RNAP II (Alarcon, 2009).

Precedent exists for the ability of CDK8 to phosphorylate enhancer-binding transcription factors. The CDK8 ortholog Srb10 in budding yeast phosphorylates Gcn4 marking this transcriptional activator of amino acid biosynthesis for recognition by the SCF(Cdc4) ubiquitin ligase. In mammalian cells, CDK8 phosphorylates the ICD signal transduction component of Notch, targeting it to the Fbw7/Sel10 ubiquitin ligase. However, whereas CDK8-mediated phosphorylation inhibits Gcn4 and Notch activity, this study shows that phosphorylation of agonist-activated Smads by CDK8/9 enables Smad-dependent transcription before triggering Smad turnover (Alarcon, 2009).

Activated Smads undergo proteasome-mediated degradation as well as phosphatase-mediated tail dephosphorylation to keep signal transduction closely tied to receptor activation. This study shows that BMP-induced Smad1-ALP generates binding sites for Smurf1, accomplishing in the nucleus what MAPK-mediated phosphorylation of basal-state Smad1 accomplishes in the cytoplasm. Similarly, TGF-β-induced linker phosphorylation of Smad2/3 provides a binding site for Nedd4L (Alarcon, 2009).

The results also reveal a positive role for ALP in Smad-dependent transcription. Smad proteins with phosphorylation-resistant linker mutations are more stable as receptor-activated signal transducers than their wild-type counterparts, yet they are transcriptionally less active. Indeed, mutation of Smad1 linker phosphorylation sites (in a wild-type Smad5 background) does not result in a straight BMP gain-of-function phenotype but rather in an unforeseen gastric epithelial phenotype. Although the interpretation of this phenotype is confounded by the contribution of MAPK signaling to linker phosphorylation, it is consistent with the present evidence that Smad1 linker phosphorylation plays an active role in BMP signaling (Alarcon, 2009).

Focusing on Smad1 to define this dual role, it was found that the phosphorylated linker sites, together with a neighboring PY motif, are recognized also by the transcriptional coactivator YAP. Smurf1 and YAP present closely related WW domains with a similar selectivity toward linker-phosphorylated Smad1. YAP is recruited with Smad1 to BMP responsive enhancers and knockdown of YAP inhibits BMP-induced Id gene responses in mouse embryonic stem cells. Both BMP and YAP act as suppressors of neural differentiation in specific contexts. This study shows that YAP supports the ability of BMP to block neural lineage commitment through the induction of Id family members, creating a link between YAP-dependent BMP transcriptional output and ES cell fate determination (Alarcon, 2009).

Thus, a common structure fulfills two opposite functions -- Smad1 transcriptional action and turnover -- by recruiting different proteins, YAP and Smurf1, at different stages of the signal transduction cycle. The cyclic recruitment and continuous turnover of transcription factors on target enhancers are required for the proper response of cells to developmental and homeostatic cues. It is proposed that Smad activation by TGF-β family agonists accomplishes this important requirement through linker phosphorylation that triggers transcriptional action and messenger turnover in one stroke (Alarcon, 2009).

Activation of the Hippo pathway by cell density cues triggers a kinase cascade that culminates in the inactivation of YAP (Yorkie in Drosophila), a transcriptional coactivator that acts through interactions with enhancer-binding factors, including TEAD/scalloped, Runx, p73, and others. Yorkie/YAP promotes cell proliferation and survival and organ growth, whereas the upstream components of the inhibitory kinase cascade constrain organ size and act as tumor suppressors. Elucidating the links between the Hippo pathway and other signaling cascades is an important open question. The evidence that YAP is recruited to BMP-activated Smad1 reveals a link between the BMP and the Hippo pathways. Both these signaling cascades have the capacity to control organ size and do so in a manner suggestive of interactions with other patterned signals. An example is the regulation of imaginal disc growth by Dpp via cell competition, a process by which slow proliferating cells are eliminated in favor of their higher-proliferating neighbors. A genetic screen for negative regulators of Dpp signaling that protect cells from being outcompeted identified upstream components of the Hippo pathway. Inactivation of these factors elevated Dpp target gene expression, presumably by failing to inhibit Yorkie, and allowed cells to outcompete their neighbors, suggesting a functional convergence of the Hippo and BMP pathways that foreshadowed the current findings (Alarcon, 2009).

Although ALP is a general event in Smad activation, YAP may not be a universal partner of linker-phosphorylated Smad1. Smad ALP likely plays a wider role potentially acting to recruit coactivators other than YAP, depending on the cellular context or the target gene. Also of interest is the identity of factors that may play an analogous role in linker-phosphorylated Smad2/3 in the TGF-β pathway. The linker phosphorylation sites and PY motifs of Smad1 and Smad2/3 are conserved in the otherwise divergent linker regions of the Drosophila orthologs Mad/dSmad1 and dSmad2, respectively. Although the contribution of the MAPK pathway in linker phosphorylation precludes a clearcut genetic investigation of these functions, they are probably conserved across metazoans. A concerted search for Smad phospholinker interacting factors would answer many of these questions and would fully elucidate the role of the Smad linker region as a centerpiece in the function, regulation, and connectivity of Smad transcription factors (Alarcon, 2009).

Search PubMed for articles about Drosophila Cdk8

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van de Peppel, J., et al. (2005). Mediator expression profiling epistasis reveals a signal transduction pathway with antagonistic submodules and highly specific downstream targets. Mol. Cell 19: 511-522. PubMed Citation: 16109375

Vincent, O., et al. (2001). Interaction of the Srb10 kinase with Sip4, a transcriptional activator of gluconeogenic genes in Saccharomyces cerevisiae. Mol. Cell. Biol. 21: 5790-5796. PubMed Citation: 11486018

Wang, G., et al. (2005). Mediator requirement for both recruitment and postrecruitment steps in transcription initiation. Mol. Cell 17: 683-694. PubMed Citation: 15749018

Yang, F., DeBeaumont, R., Zhou, S. and Naar, A. M. (2004). The activator-recruited cofactor/Mediator coactivator subunit ARC92 is a functionally important target of the VP16 transcriptional activator. Proc. Natl. Acad. Sci. 101: 2339-2344. PubMed Citation: 14983011

Yang, F., et al. (2006). An ARC/Mediator subunit required for SREBP control of cholesterol and lipid homeostasis. Nature 442: 700-704. PubMed Citation: 16799563

Yoda, A., Kouike, H., Okano, H. and Sawa, H. (2005). Components of the transcriptional Mediator complex are required for asymmetric cell division in C. elegans. Development 132: 1885-1893. PubMed Citation: 15790964

Yuan, C. X., et al. (1998). The TRAP220 component of a thyroid hormone receptor-associated protein (TRAP) coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion. Proc. Natl. Acad. Sci. 95: 7939-7944. PubMed Citation: 9653119

Zhang, H. and Emmons, S. W. (2000). A C.elegans mediator protein confers regulatory selectivity on lineage- specific expression of a transcription factor gene. Genes Dev. 14: 2161-2172. PubMed Citation: 10970880

date revised: 28 December 2011

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