InteractiveFly: GeneBrief

Cyclin-dependent kinase 8: Biological Overview | References

Gene name - Cyclin-dependent kinase 8

Synonyms -

Cytological map position - 67C10-67C10

Function - signaling

Keywords - Mediator complex, leg, eye, phosphorylation of PolII

Symbol - Cdk8

FlyBase ID: FBgn0015618

Genetic map position - 3L: 9,830,185..9,831,750 [-]

Classification - serine/threonine kinase

Cellular location - nuclear

NCBI links: EntrezGene

Cdk8 orthologs: Biolitmine
Recent literature
Gao, X., Xie, X. J., Hsu, F. N., Li, X., Liu, M., Hemba-Waduge, R. U., Xu, W. and Ji, J. Y. (2018). CDK8 mediates the dietary effects on developmental transition in Drosophila. Dev Biol. PubMed ID: 30352217
The complex interplay between genetic and environmental factors, such as diet and lifestyle, defines the initiation and progression of multifactorial diseases, including cancer, cardiovascular and metabolic diseases, and neurological disorders. Given that most of the studies have been performed in controlled experimental settings to ensure the consistency and reproducibility, the impacts of environmental factors, such as dietary perturbation, on the development of animals with different genotypes and the pathogenesis of these diseases remain poorly understood. By analyzing the cdk8 and cyclin C (cycC) mutant larvae in Drosophila, it was previously reported that the CDK8-CycC complex coordinately regulates lipogenesis by repressing dSREBP (sterol regulatory element-binding protein)-activated transcription and developmental timing by activating EcR (ecdysone receptor)-dependent gene expression. This study reports that dietary nutrients, particularly proteins and carbohydrates, modulate the developmental timing through the CDK8/CycC/EcR pathway. cdk8 and cycC mutants are sensitive to the levels of dietary proteins and seven amino acids (arginine, glutamine, isoleucine, leucine, methionine, threonine, and valine). Those mutants are also sensitive to dietary carbohydrates, and they are more sensitive to monosaccharides than disaccharides. These results suggest that CDK8-CycC mediates the dietary effects on metabolism and developmental timing in Drosophila larvae.
Chung, H. L., Mao, X., Wang, H., Park, Y. J., Marcogliese, P. C., Rosenfeld, J. A., Burrage, L. C., Liu, P., Murdock, D. R., Yamamoto, S., Wangler, M. F., Chao, H. T., Long, H., Feng, L., Bacino, C. A., Bellen, H. J. and Xiao, B. (2020). De Novo Variants in CDK19 Are Associated with a Syndrome Involving Intellectual Disability and Epileptic Encephalopathy. Am J Hum Genet. PubMed ID: 32330417
Three unrelated individuals were identified with de novo missense variants in CDK19, encoding a cyclin-dependent kinase protein family member that predominantly regulates gene transcription. These individuals presented with hypotonia, global developmental delay, epileptic encephalopathy, and dysmorphic features. CDK19 is conserved between vertebrate and invertebrate model organisms, but currently abnormalities in CDK19 are not known to be associated with a human disorder. Loss of Cdk8, the fly homolog of CDK19, causes larval lethality, which is suppressed by expression of human CDK19 reference cDNA. In contrast, the CDK19 p.Tyr32His and p.Thr196Ala variants identified in the affected individuals fail to rescue the loss of Cdk8 and behave as null alleles. Additionally, neuronal RNAi-mediated knockdown of Cdk8 in flies results in semi-lethality. The few eclosing flies exhibit severe seizures and a reduced lifespan. Both phenotypes are fully suppressed by moderate expression of the CDK19 reference cDNA but not by expression of the two variants. Finally, loss of Cdk8 causes an obvious loss of boutons and synapses at larval neuromuscular junctions (NMJs). Together, these findings demonstrate that human CDK19 fully replaces the function of Cdk8 in the fly, the human disease-associated CDK19 variants behave as strong loss-of-function variants, and deleterious CDK19 variants underlie a syndromic neurodevelopmental disorder.
Xu, W., Xie, X. J., Faust, A. K., Liu, M., Li, X., Chen, F., Naquin, A. A., Walton, A. C., Kishbaugh, P. W. and Ji, J. Y. (2020). All-Atomic Molecular Dynamic Studies of Human and Drosophila CDK8: Insights into Their Kinase Domains, the LXXLL Motifs, and Drug Binding Site. Int J Mol Sci 21(20). PubMed ID: 33053834
Cyclin-dependent kinase 8 (CDK8) and its regulatory partner Cyclin C (CycC) play conserved roles in modulating RNA polymerase II (Pol II)-dependent gene expression. To understand the structure and function relations of CDK8, the structures of human and Drosophila CDK8 proteins were analyzed using molecular dynamics simulations, combined with functional analyses in Drosophila. Specifically, the structural differences were evaluated between hCDK8 and dCDK8 to predict the effects of the LXXLL motif mutation (AQKAA), the P154L mutations, and drug binding on local structures of the CDK8 proteins. First, it was observed that both the LXXLL motif and the kinase activity of CDK8 are required for the normal larval-to-pupal transition in Drosophila. Second, molecular dynamic analyses have revealed that hCDK8 has higher hydrogen bond occupation of His149-Asp151 and Asp151-Asn156 than dCDK8. Third, the substructure of Asp282, Phe283, Arg285, Thr287 and Cys291 can distinguish human and Drosophila CDK8 structures. In addition, there are two hydrogen bonds in the LXXLL motif: a lower occupation between L312 and L315, and a relatively higher occupation between L312 and L316. Human CDK8 has higher hydrogen bond occupation between L312 and L316 than dCDK8. Moreover, L312, L315 and L316 in the LXXLL motif of CDK8 have the specific pattern of hydrogen bonds and geometries, which could be crucial for the binding to nuclear receptors. Furthermore, the P154L mutation dramatically decreases the hydrogen bond between L312 and L315 in hCDK8, but not in dCDK8. The mutations of P154L and AQKAA modestly alter the local structures around residues 154. Finally, the inhibitor-induced conformational changes of hCDK8 were identified, and the results suggest a structural difference in the drug-binding site between hCDK8 and dCDK8. Taken together, these results provide the structural insights into the roles of the LXXLL motif and the kinase activity of CDK8 in vivo.

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, Cdk8K185, 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 Cdk8K185 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, CycCY5, deletes the entire P insert plus 1429 and 1304 bp of 5'- and 3'-flanking genomic DNA, respectively. Thus, CycCY5 removes all CycC protein-coding sequences plus parts of its overlapping 5'- and 3'-gene neighbors, CG3731 and CG3641. CycCY5 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. CycCY5 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 Cdk8K185 and CycCY5 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 Med12T241 and Med13T606 (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 Nintra, 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).

Mad linker phosphorylations control the intensity and range of the BMP-activity gradient in developing Drosophila tissues

The BMP ligand Dpp, operates as a long range morphogen to control many important functions during Drosophila development from tissue patterning to growth. The BMP signal is transduced intracellularly via C-terminal phosphorylation of the BMP transcription factor Mad, which forms an activity gradient in developing embryonic tissues. This study shows that Cyclin dependent kinase 8 and Shaggy phosphorylate three Mad linker serines. Linker phosphorylations control the peak intensity and range of the BMP signal across rapidly developing embryonic tissues. Shaggy knockdown broadened the range of the BMP-activity gradient and increased high threshold target gene expression in the early embryo, while expression of a Mad linker mutant in the wing disc resulted in enhanced levels of C-terminally phosphorylated Mad, a 30% increase in wing tissue, and elevated BMP target genes. In conclusion, these results describe how Mad linker phosphorylations work to control the peak intensity and range of the BMP signal in rapidly developing Drosophila tissues (Aleman, 2014: PubMed).

CDK8-Cyclin C mediates nutritional regulation of developmental transitions through the Ecdysone receptor in Drosophila

EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition (Xie, 2015).

In animals, the amount of juvenile growth is controlled by the coordinated timing of maturation and growth rate, which are strongly influenced by the environmental factors such as nutrient availability. This is particularly evident in arthropods, such as insects, arachnids and crustaceans, which account for over 80% of all described animal species on earth. Characterized by their jointed limbs and exoskeletons, juvenile arthropods have to replace their rigid cuticles periodically by molting. In insects, the larval-larval and larval-pupal transitions are controlled by the interplay between juvenile hormone (JH) and steroid hormone ecdysone. Drosophila has been a powerful system for deciphering the conserved mechanisms that regulate hormone signaling, sugar and lipid homeostasis, and the molecular mechanisms underlying the nutritional regulation of development. In Drosophila, all growth occurs during the larval stage when larvae constantly feed, and as a result their body mass increases approximately 200-fold within 4 d, largely due to de novo lipogenesis. At the end of the third instar, pulses of ecdysone, combined with a low level of JH, trigger the larval-pupal transition and metamorphosis. During this transition, feeding is inhibited, and after pupariation, feeding is impossible, thus the larval-pupal transition marks when energy metabolism is switched from energy storage by lipogenesis in larvae to energy utilization by lipolysis in pupae (Xie, 2015).

The molecular mechanisms of ecdysone-regulated metamorphosis and developmental timing have been studied extensively in Drosophila. Ecdysone binds to the Ecdysone Receptor (EcR), which heterodimerizes with Ultraspiracle (USP), an ortholog of the vertebrate Retinoid X Receptor (RXR). By activating the expression of genes whose products are required for metamorphosis, ecdysone and EcR-USP are essential for the reorganization of flies' body plans before emerging from pupal cases as adults. Despite the tremendous progress in understanding of the physiological and developmental effects of EcR-USP signaling, the molecular mechanism of how the EcR-USP transcription factor interacts with the general transcription machinery of RNA polymerase II (Pol II) and stimulates its target gene expression remains mysterious. EcR is colocalized with Pol II in Bradysia hygida and Chironomus tentans. Although a number of proteins, such as Alien, Bonus, Diabetes and Obesity Regulated (dDOR), dDEK, Hsc70, Hsp90, Rigor mortis (Rig), Smrter (Smr), Taiman, and Trithorax-related (TRR), have been identified as regulators or cofactors of EcR-mediated gene expression, it is unknown how these proteins communicate with the general transcription machinery and whether additional cofactors are involved in EcR-mediated gene expression. In addition, it remains poorly understood how EcR activates transcription correctly after integrating nutritional and developmental cues (Xie, 2015).

The multisubunit Mediator complex serves as a molecular bridge between transcriptional factors and the core transcriptional machinery, and is thought to regulate most (if not all) of Pol II-dependent transcription. Biochemical analyses have identified two major forms of the Mediator complexes: the large and the small Mediator complexes. In addition to a separable 'CDK8 submodule', the large Mediator complex contains all but one (MED26) of the subunits of the small Mediator complex. The CDK8 submodule is composed of MED12, MED13, CDK8, and CycC. CDK8 is the only enzymatic subunit of the Mediator complex, and CDK8 can both activate and repress transcription depending on the transcription factors with which it interacts. Amplification and mutation of genes encoding CDK8, CycC, and other subunits of Mediator complex have been identified in a variety of human cancers, however, the function and regulation of CDK8-CycC in non-disease conditions remain poorly understood. CDK8 and CycC are highly conserved in eukaryotes, thus analysis of the functional regulation of CDK8-CycC in Drosophila is a viable approach to understand their activities (Xie, 2015).

Previous, work has shown that CDK8-CycC negatively regulates the stability of sterol regulatory element-binding proteins (SREBPs) by directly phosphorylating a conserved threonine residue. This study now reports that CDK8-CycC also regulates developmental timing in Drosophila by linking nutrient intake with EcR-activated gene expression. Homozygous cdk8 or cycC mutants resemble EcR mutants in both pupal morphology and retarded developmental transitions. Despite the elevation of both EcR and USP proteins in cdk8 or cycC mutants, genome-wide gene expression profiling analyses reveal systematic down-regulation of EcR-target genes, suggesting the CDK8-CycC defect lies between the receptor complex and transcriptional activation. CDK8-CycC is required for EcR-USP transcription factor binding to EcR target genes. Mass spectrometry analysis for proteins that co-immunoprecipitate with EcR and USP has identified multiple Mediator subunits, including CDK8 and CycC, and yeast two-hybrid assays have revealed that CDK8 and Med14 can directly interact with the EcR-AF1 domain. Furthermore, the dynamic changes of CDK8, EcR, USP, and SREBP correlated with the fundamental roles of SREBP in regulating lipogenesis and EcR-USP in regulating metamorphosis during the larval–pupal transition. Importantly, it was shown that starving the early third instar larvae causes precocious increase of CDK8, EcR and USP proteins, as well as premature inactivation of SREBP; whereas refeeding of the starved larvae reduces CDK8, EcR, and USP proteins, but potently stimulates SREBP activity. These results suggest a dual role of CDK8-CycC, linking nutrient intake to de novo lipogenesis (by inhibiting SREBP) and developmental signaling (by regulating EcR-dependent transcription) during the larval–pupal transition (Xie, 2015).

Through EcR-USP, ecdysone plays pivotal roles in controlling developmental timing in Drosophila. This study shows that cdk8 or cycC mutants resemble EcR-B1 mutants and CDK8-CycC is required for proper activation of EcR-target genes. Molecular and biochemical analyses suggest that CDK8-CycC and the Mediator complexes are directly involved in EcR-dependent gene activation. In addition, the protein levels of CDK8 and CycC are up-regulated at the onset of the wandering stage, closely correlated with the activation of EcR-USP and down-regulation of SREBP-dependent lipogenesis during the larval–pupal transition. Remarkably, starvation of the feeding larvae leads to premature up-regulation of CDK8 and EcR-USP, and precocious down-regulation of SREBP, while refeeding of the starved larvae results in opposite effects on the CDK8-SREBP/EcR network. Thus, it is proposed that CDK8-CycC serves as a key mediator linking food consumption and nutrient intake to EcR-dependent developmental timing and SREBP-dependent lipogenesis during the larval–pupal transition (Xie, 2015).

The Mediator complex is composed of up to 30 different subunits, and biochemical analyses of the Mediator have identified the small Mediator complex and the large Mediator complex, with the CDK8 submodule being the major difference between the two complexes. Several reports link EcR and certain subunits of the Mediator complex. For example, Med12 and Med24 were shown to be required for ecdysone-triggered apoptosis in Drosophila salivary glands. It was recently reported that ecdysone and multiple Mediator subunits could regulate cell-cycle exit in neuronal stem cells by changing energy metabolism in Drosophila, and specifically, EcR was shown to co-immunoprecipitate with Med27. However, exactly how Mediator complexes are involved in regulating EcR-dependent transcription remains unknown. The current data suggest that CDK8 and CycC are required for EcR-activated gene expression. Loss of either CDK8 or CycC reduced USP binding to EcR target promoters, diminished EcR target gene expression, and delayed developmental transition, which are reminiscent of EcR-B1 mutants. Importantly, mass spectrometry analysis for proteins that co-immunoprecipitate with EcR or USP has identified multiple Mediator subunits, including all four subunits of the CDK8 submodule (Xie, 2015).

Taken together, previous works and the present work highlight a critical role of the Mediator complexes including CDK8-CycC in regulating EcR-dependent transcription. How does CDK8-CycC regulate EcR-activated gene expression? Biochemical analyses show that CDK8 can interact with EcR and USP in vivo and that CDK8 can directly interact with EcR-AF1. These observations, together with the current understanding of how nuclear receptors and Mediator coordinately regulate transcription, suggest that CDK8-CycC may positively and directly regulate EcR-dependent transcription. Yeast two-hybrid analysis indicates that the recruitment of CDK8-CycC to EcR-USP can occur via interactions between CDK8 and the AF1 domain of EcR. Interestingly, this assay also revealed a direct interaction between EcR-AF1 and a fragment of Med14 that contains the LXXLL motif. In future work, it will be interesting to determine whether CDK8 and Med14 compete with each other in binding with the EcR-AF1, whether they interact with EcR-AF1 sequentially in activating EcR-dependent transcription, and how the Mediator complexes coordinate with other known EcR cofactors in regulating EcR-dependent gene expression (Xie, 2015).

In cdk8 or cycC mutants, the binding of USP to the promoters of the EcR target genes is significantly compromised, even though nuclear protein levels of both EcR and USP are increased. It is unclear how CDK8-CycC positively regulates EcR-USP binding to EcREs near promoters. CDK8 can directly phosphorylate a number of transcription factors, such as Notch intracellular domain, E2F1, SMADs, SREBP, STAT1, and p53. Interestingly, the endogenous EcR and USP are phosphorylated at multiple serine residues, and treatment with 20E enhances the phosphorylation of USP. Protein kinase C has also been proposed to phosphorylate USP. It will be interesting to determine whether CDK8 can also directly phosphorylate either EcR or USP, thereby potentiating expression of EcR target genes and integrating signals from multiple signaling pathways (Xie, 2015).

Although a direct role for CDK8-CycC to regulate EcR-USP activated gene expression is favored, it was not possible to exclude the potential contribution of impaired biosynthesis of 20E to the developmental defects in cdk8 or cycC mutants. For example, the expression of genes involved in synthesis of 20E, such as sad and spok, is significantly reduced in cdk8 or cycC mutant larvae. Indeed, the ecdysteroid titer is significantly lower in cdk8 mutants than control from the early L3 to the WPP stages, and feeding the cdk8 mutant larvae with 20E can partially reduce the retardation in pupariation. Nevertheless, impaired ecdysone biosynthesis alone cannot explain developmental defects that were characterized in this report for the following reasons. First, feeding cdk8 or cycC mutants with 20E cannot rescue the defects in pupal morphology, developmental delay, and the onset of pupariation. Second, the expression of EcRE-lacZ reporter in cdk8 or cycC mutant salivary glands cannot be as effectively stimulated by 20E treatment as in control. Third, knocking down of either cdk8 or cycC in PG did not lead to obvious defects in developmental timing. Therefore, the most likely scenario is that the cdk8 or cycC mutants are impaired not only in 20E biosynthesis in the PG, but also in EcR-activated gene expression in peripheral tissues. Defects in either ecdysone biosynthesis or EcR transcriptional activity will generate the same outcome: diminished expression of the EcR target genes, thereby delayed onset of pupariation (Xie, 2015).

How CDK8-CycC regulates biosynthesis of ecdysone in PG remains unknown. Several signaling pathways have been proposed to regulate ecdysone biosynthesis in Drosophila PG, including PTTH and Drosophila insulin-like peptides (dILPs)-activated receptor tyrosine kinase pathway and Activins/TGFβ signaling pathway. Interestingly, CDK8 has been reported to regulate the transcriptional activity of SMADs, transcription factors downstream of the TGFβ signaling pathway, in both Drosophila and mammalian cells. Thus, it is conceivable that the effect of cdk8 or cycC mutation on ecdysone biosynthesis may due to dysregulated TGFβ signaling in the PG (Xie, 2015).

An effort to explore the potential role of food consumption and nutrient intake on CDK8-CycC has resulted an unexpected observation that the protein level of CDK8 is strongly influenced by starvation and refeeding: starvation potently increased CDK8 level, while refeeding has opposite effect, and both occur post-transcriptionally. The importance of this observation is highlighted in two aspects. First, considering the generally repressive role of CDK8 on Pol II-dependent gene expression, up-regulation of CDK8 may provide an efficient way to quickly tune down most of the Pol II-dependent transcription in response to starvation; while down-regulation of CDK8 in response to refeeding may allow many genes to express when nutrients are abundant. Second, it will be necessary to test whether the effects of nutrient intake on CDK8-CycC is conserved in mammals. If so, considering that both CDK8 and CycC are dysregulated in a variety of human cancers, the effects of nutrient intake on CDK8 may have important implications in not only understanding of the effects of nutrients on tumorigenesis, but also providing nutritional guidance for patients with cancer (Xie, 2015).

Major dietary components including carbohydrates, lipids, and proteins, can strongly influence the developmental timing in Drosophila. Excessive dietary carbohydrates repress growth and potently retard the onset of pupariation. One elegant model proposed to explain how high sugar diet delays developmental timing is that high sugar diet reduces the activity of the Target of Rapamycin (TOR) in the PG, thereby reducing the secretion of ecdysone and delaying the developmental transition. Previously, it was reported that insulin signaling could down-regulate CDK8-CycC, and that ectopic expression of CycC could antagonize the effect of insulin stimulation in mammalian cells, as well as the effect of refeeding on the expression of dFAS in Drosophila (Zhao, 2012). Although the mRNA levels of TOR and insulin receptor (InR) are not significantly affected in cdk8 or cycC mutants, it is necessary to further study whether and how different dietary components may regulate CDK8-CycC in the future (Xie, 2015).

Developmental genetic analyses of the cdk8 and cycC mutants have revealed major defects in fat metabolism and developmental timing. De novo lipogenesis, which is stimulated by insulin signaling, contributes significantly to the rapid increase of body mass during the constant feeding larval stage. This process is terminated by pulses of ecdysone that trigger the wandering behavior at the end of the L3 stage, followed by the onset of the pupariation. Insulin and ecdysone signaling are known to antagonize each other, and together determine body size of Drosophila. The genetic interaction is established, but the detailed molecular mechanisms are not. The SREBP family of transcription factors controls the expression of lipogenic enzymes in metazoans and the expression of cholesterogenic enzymes in vertebrates. Previous work shows that CDK8 directly phosphorylates the nuclear SREBP proteins on a conserved threonine residue and promotes the degradation of nuclear SREBP proteins. Consistent with the lipogenic role of SREBP and the inhibitory role of insulin to CDK8-CycC, the transcriptional activity of SREBP is high while the levels of CDK8-CycC and EcR-USP are low prior to the onset of wandering stage. Subsequently during the wandering and non-mobile, non-feeding pupal stage, the transcriptional activity of SREBP is dramatically reduced, accompanied by the significant accumulation of CDK8-CycC and EcR-USP (Xie, 2015).

The causal relationship of these phenomena was further tested by starvation and refeeding experiments. On the one hand, it was observed that the levels of CDK8, EcR and USP are potently induced by starvation, while the mature SREBP level and the transcriptional activity of SREBP are reduced by starvation. Starvation of larvae prior to the two nutritional checkpoints in early L3, known as minimum viable weight and critical weight, which are reached almost simultaneously in Drosophila, will lead to larval lethality; while starvation after larvae reach the critical weight will lead to early onset of pupariation and formation of small pupae. Thus, this nutritional checkpoint ensures the larvae have accumulated sufficient growth before metamorphosis initiation. If the status with high CDK8, EcR, and USP is regarded as an older or later stage, these results indicate that starvation shifts the regulatory network precociously (see Model for the CDK8-SREBP/EcR regulatory network). On the other hand, the current analyses of refed larvae show that refeeding potently reduced the levels of CDK8, EcR and USP. If the status with low CDK8, EcR, and USP is considered as a younger or earlier stage, these results indicate that refeeding delays the activation of this network, which is consistent with the model and delayed pupariation as observed. Taken together, these results based on starved and refed larvae suggest that CDK8-CycC is a key regulatory node linking nutritional cues with de novo lipogenesis and developmental timing (Xie, 2015).

The larval-pupal transition is complex and dynamic. Although the expression of SREBP target genes fit well with the predicted effects of starvation and refeeding, the expression of EcR targets during the stage that was analyzed in this study does not reflect the changes in the protein levels of EcR and USP. It is reasonable to consider that CDK8-CycC and EcR-USP are necessary, but not sufficient, for the activation of EcR target genes. One possibility is that there is a delay on synthesis of 20E or other cofactors that are required for EcR-activated gene expression in response to starvation. Indeed, the 20E levels were measured during the first 16 hr of starvation, and no significant difference was observed between fed and starved larvae. It will be necessary to further analyze the effect of starvation on 20E synthesis at later time points in the future (Xie, 2015).

Taken together, a model is proposed whereby CDK8-CycC functions as a regulatory node that coordinates de novo lipogenesis during larval stage and EcR-dependent pupariation in response to nutritional cues. It is likely that pulses of 20E synthesized in the PG, and subsequent behavioral change from feeding to wandering, ultimately trigger the transition from SREBP-dependent lipogenesis to EcR-dependent pupariation. The opposite effects of CDK8-CycC on SREBP- and EcR-dependent gene expression suggest that the role of CDK8 on transcription is context-dependent (Xie, 2015).

In conclusion, this study illustrates how CDK8-CycC regulates EcR-USP-dependent gene expression, and the results suggest that CDK8-CycC may function as a regulatory node linking fat metabolism and developmental timing with nutritional cues during Drosophila development (Xie, 2015).

De Novo Variants in CDK19 Are Associated with a Syndrome Involving Intellectual Disability and Epileptic Encephalopathy

Three unrelated individuals were identified with de novo missense variants in CDK19, encoding a cyclin-dependent kinase protein family member that predominantly regulates gene transcription. These individuals presented with hypotonia, global developmental delay, epileptic encephalopathy, and dysmorphic features. CDK19 is conserved between vertebrate and invertebrate model organisms, but currently abnormalities in CDK19 are not known to be associated with a human disorder. Loss of Cdk8, the fly homolog of CDK19, causes larval lethality, which is suppressed by expression of human CDK19 reference cDNA. In contrast, the CDK19 p.Tyr32His and p.Thr196Ala variants identified in the affected individuals fail to rescue the loss of Cdk8 and behave as null alleles. Additionally, neuronal RNAi-mediated knockdown of Cdk8 in flies results in semi-lethality. The few eclosing flies exhibit severe seizures and a reduced lifespan. Both phenotypes are fully suppressed by moderate expression of the CDK19 reference cDNA but not by expression of the two variants. Finally, loss of Cdk8 causes an obvious loss of boutons and synapses at larval neuromuscular junctions (NMJs). Together, these findings demonstrate that human CDK19 fully replaces the function of Cdk8 in the fly, the human disease-associated CDK19 variants behave as strong loss-of-function variants, and deleterious CDK19 variants underlie a syndromic neurodevelopmental disorder (Chung, 2020).

Enhancer architecture sensitizes cell-specific responses to Notch gene dose via a bind and discard mechanism

Notch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, there is only a limited mechanistic understanding of phenotype variability due to gene dosage. This study unexpectedly found that inserting an enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can induce a subset of Notch haploinsufficiency phenotypes in Drosophila with wild type Notch gene dose. Using Drosophila genetics, it was shown that this enhancer induces Notch phenotypes in a Cdk8-dependent, transcription-independent manner. Mathematical modeling was combined with quantitative trait and expression analysis to build a model that describes how changes in Notch signal production versus degradation differentially impact cellular outcomes that require long versus short signal duration. Altogether, these findings support a 'bind and discard' mechanism in which enhancers with specific binding sites promote rapid Cdk8-dependent Notch turnover, and thereby reduce Notch-dependent transcription at other loci and sensitize tissues to gene dose based upon signal duration (Kuang, 2020).

Haploinsufficiency, or the inability to complete a cellular process with one functional allele of a given gene, manifests in tissue and organ defects with variable penetrance and severity. For example, Notch (N) haploinsufficiency, which was discovered in Drosophila, causes a variety of tissue-specific defects including wing notching and extra sensory bristle formation that can vary greatly in penetrance and expressivity. Notch pathway haploinsufficiency was subsequently observed in mammals, as Notch1 heterozygous mice have heart valve and endothelium defects, whereas Notch2 heterozygotes have defects in bone, kidney and marginal zone B cells. A single allele of NOTCH2 or the JAG1 ligand can also cause pathological phenotypes in humans, as heterozygosity of either gene can result in a variably penetrant developmental syndrome known as Alagille. Thus, Notch gene dose sensitivity has been observed in a variety of Notch-dependent tissues in both humans and animals. Unfortunately, a mechanistic understanding of what causes some tissues to be highly sensitive to Notch gene dose and what factors impact the variable penetrance and severity of Notch haploinsufficiency phenotypes is lacking (Kuang, 2020).

Molecularly, Notch signaling is initiated by ligand-induced proteolysis of the Notch receptor to release the Notch intracellular domain (NICD) from the membrane. NICD subsequently transits into the nucleus, binds to the Cbf1/Su(H)/Lag1 (CSL) transcription factor (TF) and the adaptor protein Mastermind (Mam), and induces gene expression via two types of DNA binding sites: independent CSL sites that bind monomeric NICD/CSL/Mam (NCM) complexes, and Su(H) paired sites (SPS) that are oriented in a head-to-head manner to promote cooperative binding between two NCM complexes. Once bound to an enhancer, the NCM complex activates transcription of associated genes via the P300 co-activator. Thus, the production of NICD is converted into changes in gene expression that ultimately regulate cellular processes during development (Kuang, 2020).

Haploinsufficiency of Notch receptor and ligand encoding genes suggests that decreased gene dosage results in a sufficiently large decrease in NICD production to cause phenotypes in a subset of tissues. There is also growing evidence that genetic changes that reduce NICD degradation can alter signal strength with pathological consequences in specific cell types. In the mammalian blood system, for example, Notch1 mutations that remove an NICD degron sequence have been associated with increased NICD levels and the development of T-cell Acute Lymphoblastic Leukemia (T-ALL) in mice and humans. Intriguingly, NICD turnover via this degron sequence has been directly linked to transcription activation, as the Mam protein interacts with the Cdk8 kinase module (CKM) of the Mediator complex, which can phosphorylate NICD to promote its ubiquitylation by the Fbxw7 E3-ligase and degradation by the proteasome. Accordingly, gene mutations that lower CKM activity have also been associated with increased NICD levels and T-ALL initiation and progression. Thus, perturbations in mechanisms that regulate either NICD production or degradation can induce cell and/or tissue specific phenotypes (Kuang, 2020).

This study used Drosophila genetics, quantitative trait and expression analysis, and mathematical modeling to unravel a unique regulatory mechanism that impacts Notch signal strength in a tissue-specific manner. First, it was unexpectedly found that an enhancer containing as few as 12 Notch dimer binding sites can induce tissue-specific phenotypes via a CKM-dependent mechanism that can be uncoupled from transcription activation. Second, based on quantitative analysis and mathematical modeling, changes in NICD degradation rates are shown to be predicted to preferentially impact long duration Notch-dependent processes, whereas genetic changes in NICD production rates (i.e., Notch haploinsufficiency) affect both short and long duration processes. Collectively, these findings provide new insights into how distinct Notch-dependent cellular processes can be differentially impacted by both enhancer architecture and signal duration to induce tissue-specific Notch defects within a complex animal (Kuang, 2020).

The results show that simply increasing the number of clustered Notch dimer sites (SPS) linked to sites for a pioneer TF can cause a tissue-specific N haploinsufficiency phenotype via a Cdk8-dependent mechanism. These findings have important implications for both enhancer biology and the mechanisms regulating Notch signal strength in specific tissues. First, the proposed Cdk8-dependent mechanism links the rapid degradation of the Notch signal (NICD) with its binding to specific loci (SPSs) in a manner that can be uncoupled from transcription activation. This 'bind and discard' mechanism reveals an unexpected global link between accessible binding sites in the epigenome, such that the collective 'drain' loci can reduce Notch-dependent transcription at other loci in the same nucleus. Moreover, since the binding sites do not have to be coupled with transcription to induce a notched wing phenotype, the findings highlight the possibility that seemingly non-functional genomic binding events could impact TF metabolism in a Cdk8-dependent manner. Given that Cdk8 interacts with many genomic loci and that a previous phospho-proteomic study identified numerous transcriptional regulators are targets of CDK8/19 phosphorylation (CDK19 is a paralog of CDK8), such a mechanism may be quite general and apply to transcription regulators beyond Notch (Kuang, 2020).

While genetic and cell culture data, as well as previous phosphorylation studies, support a direct link between CKM activity and NICD degradation, it should be noted that the CKM also phosphorylates other proteins that could contribute to the differences in wing versus macrochaetae phenotypes. Intriguingly, one of the high confidence CKM targets in mammalian cells was MAML1, indicating that CKM activity may directly regulate Notch output by phosphorylating multiple components of the Notch transcription complex. In addition, CCNC (CycC), MED12, and MED13 were all found to be high confidence CDK8/19 targets, suggesting that CKM activity may directly impact the turnover of key components of its own complex. It's thus not surprising that removing an allele of cycC, kto (med12) or skd (med13) had a much larger impact on the Notch haploinsufficient wing phenotype compared to changing the gene dose of cdk8. Moreover, these genetic data are consistent with prior studies in yeast showing that structural/regulatory components of macromolecular complexes, such as CycC/Med12/Med13, are enriched in haploinsufficiency genes, whereas enzymes are generally under-represented from the list of dose sensitive genes (Kuang, 2020).

Second, the data support the idea that not all Notch binding sites are equally capable of marking NICD for degradation, and that enhancer architecture plays a key role in modulating NICD turnover. For instance, only Notch dimer but not Notch monomer sites are sufficient to generate phenotypes, and even SPS-containing enhancers differ in their ability to induce phenotypes based on the absence/presence of pioneer TF sites. Notably, this study found that enhancers with either synthetic SPS sites designed to limit additional TF input or an endogenous E(spl)m8 SPS with adjacent binding sites capable of providing negative feedback were sufficient to induce notched wing phenotypes when coupled to pioneer TF sites. Since Grh and Zld binding is sufficient to increase chromatin opening, these findings suggest enhancer accessibility alters the rate at which NICD is metabolized by Notch dimer sites. Intriguingly, ChIP-seq data for Grh and Zld reveals extensive binding to the Enhancer of Split (E(spl)) locus that contains numerous SPS-containing Notch regulated enhancers. However, it is important to note that while an SPS-containing enhancer lacking pioneer TF sites failed to induce phenotypes in wild type flies, it did significantly increase wing notching in a sensitized genetic background. These findings suggest that SPS-containing enhancers promote NICD degradation at differing rates based on the presence of nearby TF sites. While the mechanistic basis for how SPS but not CSL sites promote NICD degradation is not known, these data highlight a potential mechanism by which enhancer architecture (i.e., Notch dimer vs monomer sites) and epigenetic 'context' (i.e., accessibility due to pioneer TF binding) can fine tune the global Notch response in different tissues (Kuang, 2020).

The finding that introducing as few as 12 SPS sites into the genome can induce notched wing phenotypes raises the question of how many functional SPS sites exist in the endogenous genome. Recent studies found that about one third of direct Notch target genes (38 of 107 genes) in human T-ALL are regulated by SPS sites, and a mouse mK4 cell line has an estimated 2500 Notch dimer dependent binding sites. These findings suggest that many SPS sites are accessible across the mammalian genome. The estimated number of SPS sites within the Drosophila genome, which is an order of magnitude smaller than most mammalian genomes, remains to be determined. However, of the 154 Notch-responsive genes identified in a Drosophila wing disc-derived cell line, eight encode E(spl) genes that are clustered within a common 40 kb locus and many E(spl) genes contain one or more SPS sites. In comparison to the E(spl) locus, the 6 SPS sites within the G6S-lacZ transgene are found within ~300 bps, and thus it is possible that concentrating SPS sites might provide an avidity impact that increases the probability of a recruited NICD molecule being marked for degradation. In fact, it is estimated that the cumulative effect of the Drosophila genome is equal to ~5 highly accessible, linked SPS-GBE sites. Future studies using endogenous SPS-containing enhancers will be needed to provide a better understanding of both the role of nearby binding sites for other TFs and how concentrating SPS sites in specific loci impacts the wing notching phenotype (Kuang, 2020).

Third, it is proposed that the differential sensitivity of Notch-dependent tissues to changes in NICD degradation (i.e., SPS-GBE sites or CKM heterozygotes) or production rates (N heterozygotes) reflects the temporal requirement for Notch signal duration. An appealing aspect of this Notch signal duration model is that it predicts that any perturbation that alters NICD signal degradation will preferentially affect long-duration processes over short duration processes, whereas perturbations that impact NICD signal production will affect both long and short duration events. Moreover, the differential sensitivity of the Notch duration model to changes in production versus degradation rates may be generalizable to the study of other signaling pathways. However, it's worth noting that additional differences between the wing and macrochaetae besides signal duration may contribute to the magnitude of change in Notch signal strength in each tissue. As an example, cis-inhibition, which determines the fraction of functional Notch receptors on the cell membrane, could play a cell-specific role in modulating NICD production to a larger degree in one tissue over another. Taking this mechanism into consideration, the assumption that Notch heterozygotes reduce NICD production by 50 percent in each tissue may be over-simplified. Thus, further experiments using a system that is amenable to systematic changes in the length of Notch signal induction are needed to thoroughly test the signal duration model in multiple tissues (Kuang, 2020).

Intuitively, the duration model suggests a mechanism underlying cell-specific context that may have implications for both developmental processes and tumorigenesis. For example, mutations in the NICD PEST domain that decouple DNA binding and degradation are common in T-cell acute lymphoblastic leukaemia (T-ALL), and CycC (CCNC) functions as a haploinsufficient tumor suppressor gene in T-ALL, at least in part, by stabilizing NICD. These findings suggest that T-ALL is highly sensitive to alterations in NICD degradation. Indeed, T-ALL cells are 'addicted' to Notch and are thus dependent on a long duration signal. As ~30% of Notch target genes in T-ALL use SPS containing enhancers, the current findings provide insight into how Notch PEST truncations and CCNC heterozygotes could each promote tumorigenesis by slowing CKM-mediated NICD turnover on SPS enhancers. Future studies focused on enhancers that recruit the CKM and other Notch-dependent cellular processes will help reveal how the temporal requirements for nuclear activities contributes to both normal development and disease states (Kuang, 2020).

The Mediator CDK8-Cyclin C complex modulates Dpp signaling in Drosophila by stimulating Mad-dependent transcription

Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. To identify upstream regulators or downstream effectors of CDK8, a dominant modifier genetic screen was performed in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. 26 genomic loci were identified whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led to identification of genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFβ, or Transforming Growth Factor-β) signaling pathway. It was observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFβ signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, this analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila (Li, 2020).

To study the function and regulation of CDK8 in vivo, a genetic system was developed that yields robust readouts for the CDK8-specific activities in developing Drosophila wings. These genetic tools provide a unique opportunity to perform a dominant modifier genetic screen, allowing identification multiple components of the Dpp/TGFβ signaling pathway that can genetically interact with the CDK8-CycC complex in vivo. Subsequent genetic and cellular analyses reveal that CDK8, CycC, and six additional subunits of the Mediator complex, as well as CDK9 and Yki are required for the Mad-dependent transcription in the wing discs. In addition, CDK8 can directly interact with the linker region of Mad. These results have extended the previous biochemical and molecular analyses on how different kinases and transcription cofactors modulate the Mad/Smad-activated gene expression in the nucleus. Further mapping of specific genes uncovered by other deficiency lines may also open up the new directions to advance understanding of the conserved function and regulation of CDK8 during development (Li, 2020).

The Mediator complex functions as a molecular bridge between gene-specific transcription factors and the RNA Pol II general transcription apparatus, and diverse transactivators have been shown to interact directly with distinct Mediator subunits. However, it is unclear whether all Mediator subunits are required by different transactivators to regulate gene expression, or whether Mediator complexes composed of fewer and different combinations of Mediator subunits exist in differentiated tissues or developmental stages. Gene-specific combinations of the Mediator subunits may be required in different transcription processes, as not all Mediator subunits are simultaneously required for all transactivation process. For instance, ELK1 target gene transcription requires Med23, but lacking Med23 does not functionally affect some other ETS transcription factors, such as Ets1 and Ets2 . Similarly, Med15 is required for the expression of Dpp target genes, but does not appear to affect the expression of EGFR (epidermal growth factor receptor) and Wg targets in Drosophila (Li, 2020).

It has been previously reported that the Med15 subunit is required for the Smad2/3-Smad4 dependent transcription, as its removal from the Mediator complex abolishes the expression of Smad-target genes and disrupts Smad2/3-regulated dorsal-ventral axis formation in Xenopus embryos. Further biochemical analyses showed that increased Med15 enhances, while its depletion decreases, the transcription of Smad2/3 target genes, and that the Med15 subunit can directly bind to the MH2 domain of Smad2 or Smad3. In Drosophila, loss or reduction of Med15 reduced the expression of Dpp targets, resulting in smaller wings and disrupted vein patterning (mainly L2). It was also observed that depletion of Med15 or CDK8 reduces the expression of a Mad-target gene. These observations support the idea that CDK8 and Med15 play a conserved and positive role in regulating Mad/Smad-activated gene expression (Li, 2020).

Aside from Med15 and CDK8, it remains unclear whether other Mediator subunits are also involved in Mad/Smad-dependent transcription. This study identified six additional Mediator subunits that are required for the Mad-dependent transcription, including CycC, Med12, Med13, Med23, Med24, and Med31. Interestingly, aside from Med23 and Med24 being specific to metazoans, counterparts of the other six subunits are not essential for cell viability in the budding yeast. The similar effects of the four CKM (CDK8 kinase module) subunits on Mad-activity suggest that they may function together to stimulate Mad-dependent transcription. It is noted that depletion of seven Mediator subunits, Med7, Med8, Med14, Med16, Med17, Med21, and Med22, severely disrupts the morphology of the wing discs, making it difficult to assay their effects on the transcriptional activity of Mad in vivo. Consistently, all corresponding subunits, except Med16, are critical for cell viability in the budding yeast. In contrast, reducing expression of the 15 remaining subunits of the Drosophila Mediator complex did not significantly alter the expression of a Mad-dependent reporter. Med1 and Med25 are loosely associated to the small Mediator complex in human cell lines. A caveat for these negative results is that depleting these subunits using the existing RNAi lines may not be sufficient to affect sal-lacZ expression, even though the majority of these transgenic RNAi lines can generate severe phenotypes in the eye, wing, or both. Further analyses are necessary to validate these negative data in the future. Taken together, the results indicate that not all Mediator subunits are required for the expression of the Mad-target genes that were tested in the developing wing discs (Li, 2020).

Interestingly, Yki/YAP, which can function as a transcriptional co-factor for Mad/Smad, was also reported to associate with several subunits of the Mediator complex to drive transcription. Specifically, Med12, Med14, Med23, and Med24 were identified from a YAP IP-mass spectrometry sample in HuCCT1 cells. Med23 was also reported to regulate Yki-dependent transcription of Diap1 in wing discs. In the current study, Yki, Med12, Med23, and Med24 were also required for Mad-dependent transcription of sal-lacZ. Although the exact molecular mechanisms of how Yki interacts with certain Mediator subunits remain unclear, it is plausible that Yki may further strengthen the binding between Mad and Med15 through interactions with other subunits such as Med12, Med23, and Med24 (Li, 2020).

Based on biochemical analyses of the Smad1 phosphomutants and cell biological analyses using cultured human epidermal keratinocytes (HaCaT cells), several kinases including CDK8, CDK9, and ERK2 were shown to phosphorylate serine residues (Ser, or S) within the linker region of pSmad1 at S186, S195, S206, and S214, or the equivalent sites in pSmad2/3/5. These modifications were proposed to regulate positively Smad1-dependent transcriptional activity. Of these sites, S206 and S214 are both conserved from Drosophila to humans. In addition, studies using Xenopus embryos and cultured L cells suggest that MAPKs may phosphorylate the linker region of Smad1 (including S214) and lead to its degradation. Nevertheless, analyses with Drosophila embryos and wing discs indicate that S212 (equivalent to human pSmad1 S214) is phosphorylated by CDK8, while S204 (unique in Drosophila) and S208 (equivalent to human pSmad1 S210) are phosphorylated by Sgg/GSK3. These studies suggest the following model in explaining how Smads activate the expression of their target genes and how this process is turned off: after Smads are phosphorylated at their C-termini and translocated into the nucleus, CDK8 and CDK9 (potentially also MAPKs) act as the priming kinases to further phosphorylate pSmads in the linker region at S206 and S214. This may facilitate the interaction between pSmads and transcriptional cofactors such as YAP, stimulating the expression of Smads target genes. Overexpression of Yki in Drosophila wing disc increases the expression of the vgQE-lacZ reporter, which validates the role of Yki/YAP in activating Mad/Smad1-dependent gene expression in vivo. Subsequently, pSmads are further phosphorylated by GSK3 within the linker region at T202 and S210, which may facilitate Smad1/5 binding to E3 ubiquitin ligases such as Smurf1 and Nedd4L, causing the degradation of Smads through the ubiquitin-proteasome pathway (Li, 2020).

Although this model is still rather speculative, it serves as a conceptual framework to explain how transactivation of Smads is coupled to its degradation, similar to other transcriptional activators. It is challenging to determine whether these kinases act redundantly or sequentially for different phosphorylation sites, the exact orders of these phosphorylation events, as well as their biological consequences in vivo. Moreover, it remains unexplored whether these regulatory mechanisms are conserved during evolution. The importance of these issues is highlighted by the critical role of TGFβ signaling in regulating the normal development of metazoans and the dysregulation of this pathway in a variety of human diseases such as cancers (Li, 2020).

The precise spatiotemporal activation of the Dpp signaling pathway in the wings discs is critical for proper formation of the stereotypical vein patterns in Drosophila. This model system provides an ideal opportunity to dissect the dynamic regulation of the Mad-activated gene expression in the nucleus. Indeed, depleting CDK8 in wing discs reduces expression of the Mad-dependent sal-lacZ reporter, suggesting that CDK8 positively regulates Mad-dependent transcription. This is consistent with the effects of CDK8 on Smad1/5-dependent transcription in mammals. Depleting CDK8 does not affect the phosphorylation of Mad at its C-terminus as revealed by pMad immunostaining, nor does it affect the physical interaction between CDK8 and the linker region of Mad, supporting the idea that CDK8 may only affect subsequent phosphorylation of Mad, presumably within the linker region (Li, 2020).

Besides CDK8-CycC, depleting CDK9-CycT also decreases the expression of the sal-lacZ reporter, supporting the notion that CDK8-CycC and CDK9-CycT may play non-redundant roles in further phosphorylating pMad in the nucleus. However, no effects of depletion of CDK7 or MAPKs on sal-lacZ expression were observed, suggesting that their role in regulating the transcriptional activity of Smads may not be conserved in Drosophila. Alternatively, the two MAPK/ERK homologs, Rolled and ERK2, may act redundantly in regulating Mad-dependent transcription. Lastly, depleting Sgg/GSK3 in the dorsal compartment of the wing disc increases the size of this compartment, yet the expression level of the sal-lacZ reporter is similar to the ventral compartment. These observations are consistent with previous reports that phosphorylations of Mad/Smad in the linker regions by CDK8-CycC and Sgg/GSK3 regulate the level and range of Mad-dependent gene expression (Li, 2020).

Together with the previous reports, the data support that CDK8-CycC and CDK9-CycT may phosphorylate pMad at the linker region, which may facilitate the binding between Yki and Mad. It is speculated that this interaction may synergize the recruitment of the Mediator complex, presumably at least through the interaction between its Med15 subunit and the MH2 domain of Mad (see Model of Mad/Smad-dependent transcription activation through the CKM and the Mediator complex.). Alternatively, Yki may also facilitate the recruitment of the whole Mediator complex through its interactions with Med12, Med23, and Med24. The synergistic interactions among Mad, Yki, the Mediator complex, and RNA Pol II may be required for the optimal transcriptional activation of the Mad-target genes (Li, 2020).

One of the challenges is to illustrate the dynamic interactions between these factors and diverse protein complexes that couple the transactivation effects of Mad/Smads on gene transcription with their subsequent degradation at the molecular level. Smad3 phosphorylation strongly correlates with Med15 levels in breast and lung cancer tissues; together, they potentiate metastasis of breast cancer cells. Thus, it will be important to test whether additional Mediator subunits that were identified in Drosophila play similar roles in mammalian cells. It will also be interesting to determine whether a partial Mediator complex, composed of a subset of the Mediator subunits, exists and regulates Mad/Smad-dependent gene expression. Furthermore, detailed biochemical analyses may yield mechanistic insights into how CDK8 and Med15 act in concert in stimulating the Mad/Smad-dependent gene expression (Li, 2020).

Wing pouch-specific alteration of CDK8 activity results in two major phenotypes: disrupted vein patterns and altered size of wing blades. While the effects on wing size and cell numbers can be explained by the role of CDK8 in regulating cell proliferation through E2F1, the effects of CDK8 on vein patterning are more complex. The stereotypical wing vein patterns in adult flies are gradually defined by elaborated spatiotemporal interplays among different signaling pathways, including Dpp, EGFR, Hedgehog (Hh), Notch (N), and Wingless (Wg), in the developing wing discs. During the larval and pupal stages, these signaling pathways and their downstream transcriptional targets coordinately control the cell proliferation and differentiation of cell in different parts of the wing disc to form individual veins (Li, 2020).

It is noteworthy that varying CDK8 activities has different effects on different veins: gain of CDK8 causes the loss of the L3 and L4 veins, but the vein patterns of L2 and L5 appear thicker and more diffusive; while the ectopic veins caused by reduction of CDK8 are mainly intertwined with the L2 and L5 veins. This analyses on the genetic interactions between CDK8 and the components of the Dpp signaling pathway led to the discovery of the role of the Mediator complex in Mad-stimulated transcription of sal. However, there is a gap in understanding ohow reduced expression of sal in wing discs is linked to the vein defects in adult wings. It is known that salm and salr (spalt-related), two members of the spalt gene family that encode zinc-finger transcriptional repressors, function downstream of the Dpp signaling pathway during development of the central part of the wing. Depletion of either salm or salr alone resulted in ectopic vein formation around L2 in adult wings, yet depletion or loss of both salm or salr caused loss of vein phenotype. In addition, elimination of L2 in ventral-anterior and ectopic L5 in dorsal-posterior were observed in salm/salr clones at different region of the wing. These observations suggest that the dosage of salm and salr in wing discs does not have a linear relationship with the wing vein patterning at the adult stage (Li, 2020).

Interestingly, it is known that the CKM complex regulates the transcriptional activities of the key transcription factors of these pathways, including N-ICD downstream of N signaling and Mad/Smad proteins. In addition, Med12 (Kohtalo, or Kto in Drosophila) and Med13 (Skuld, or Skd in Drosophila) subunits of the CKM interact with Pangolin (the lymphoid-enhancing factor (LEF)/T cell factor (TCF) homolog in Drosophila), the key transcription factor downstream of Wg signaling, through the transcriptional cofactors such as Pygopus, Legless, and Armadillo. In mammalian cells, Med12 is also known to regulate the activities of Gli proteins, the key transcription factors downstream of Hh signaling. Furthermore, the Mediator subunit Med23 interacts with ETS (E-twenty six transcription factor) proteins, a family of key transcription factors downstream of the EGFR signaling pathway. However, whether CDK8-CycC also regulates TCF-, ETS- or Gli-dependent transcription is still not understood. Nevertheless, these studies in other biological contexts suggest that the effects of CDK8 on wing vein patterning are not likely solely through the Dpp signaling pathway. Therefore, it is speculated that the potential interactions between CDK8 and the aforementioned signaling pathways may contribute to these differential effects on distinct veins. Further analyses of these cross-talks, as well as further mapping of other Df lines that modify the CDK8-specific vein phenotypes, may yield the insights into the molecular and dynamic mechanisms underlying these vein phenotypes (Li, 2020).

To understand how dysregulated CDK8-CycC contributes to a variety of human cancers, it is essential to elucidate the function and regulation of CDK8 in vivo. Given that the CDK8-CycC pair and other subunits of the Mediator complex are conserved in almost all eukaryotes, Drosophila serves as an ideal model system to identify both the upstream regulators and the downstream effectors of CDK8 activity in vivo. The dominant modifier genetic screen is based on the wing vein phenotypes caused by specific alteration of CDK8 activity in the developing wing disc, which serves as a unique in vivo readout for the CDK8-specific activities in metazoans. This screen led to the identification of 26 genomic regions that include loci whose haplo-insufficiency could consistently modify CDK8-CycC depletion or CDK8-overexpression phenotypes. Identification of Dad and genes encoding additional components of the Dpp signaling pathway provides a proof of principle for this approach. Since each of the chromosomal deficiencies uncovers multiple genes, further mapping of the relevant genome regions is expected to identify the specific genetic loci encoding factors that may function either upstream or downstream of CDK8 in vivo. It is hoped that further analyses of the underlying molecular mechanisms in both Drosophila and mammalian systems will advance understanding of how dysregulation of CDK8 contributes to human diseases, thereby aiding the development of therapeutic approaches (Li, 2020).

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-VP16Delta456FP442. However, the amounts of dGCN5, dTAFII40, dTAFII250, 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 dTAFII250, 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 dTAFII250, 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-1Rep 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).

A genome-wide RNA interference screen identifies a differential role of the mediator CDK8 module subunits for GATA/ RUNX-activated transcription in Drosophila

Transcription factors of the RUNX and GATA families play key roles in the control of cell fate choice and differentiation, notably in the hematopoietic system. During Drosophila hematopoiesis, the RUNX factor Lozenge and the GATA factor Serpent cooperate to induce crystal cell differentiation. This study used Serpent/Lozenge-activated transcription as a paradigm to identify modulators of GATA/RUNX activity by a genome-wide RNA interference screen in cultured Drosophila blood cells. Among the 129 factors identified, several belong to the Mediator complex. Mediator is organized in three modules plus a regulatory "CDK8 module," composed of Med12, Med13, CycC, and Cdk8, which has long been thought to behave as a single functional entity. Interestingly, the data demonstrate that Med12 and Med13 but not CycC or Cdk8 are essential for Serpent/Lozenge-induced transactivation in cell culture. Furthermore, in vivo analysis of crystal cell development show that, while the four CDK8 module subunits control the emergence and the proliferation of this lineage, only Med12 and Med13 regulate its differentiation. It is thus proposed that Med12/Med13 acts as a coactivator for Serpent/Lozenge during crystal cell differentiation independently of CycC/Cdk8. More generally, it is suggested that the set of conserved factors identified in this study may regulate GATA/RUNX activity in mammals (Gobert, 2010).

During development, a combination of general and lineage-specific transcription factors integrate different regulatory inputs at the transcriptional levels to unfold the proper gene expression program. The identification of the complete panel of genes that participate in the regulation of the activity of these transcription factors is critical to understand the fine-tuning of transcription that underlies cellular differentiation. In this study, a genome-wide RNAi screen was conducted to uncover regulators of the activity of the GATA/RUNX complex Srp/Lz. This approach highlighted the function of the Mediator complex in Srp/Lz-induced transcriptional activation. Moreover, it was found that, within the Mediator CDK8 module, Med12 and Med13 act independently of CycC and Cdk8 to promote Srp/Lz-dependent transactivation and blood cell differentiation (Gobert, 2010).

The activity of GATA and RUNX transcription factors has been shown to be regulated by interaction with several factors, such as the coactivator CBP/p300 or the corepressors HDAC and Sin3A. However, proper transcriptional regulation relies on the coordinated action of several transcription factors binding a particular cis-regulatory element. Notably, GATA and RUNX factor have been shown to cooperate in both mammals and Drosophila to regulate the expression of specific target genes. Hence, this study used Srp/Lz cooperation as a paradigm to identify putative coregulators of GATA/RUNX activity. Among the genes that were identified, five (CKD9, SIN3A, MED1, enok homolog MYST3/MOZ, and pnt homolog ETS1) have been linked previously to GATA and/or RUNX activity in mammals, and four (Pcf11, CtBP, med13, and Sin3A) have been linked to crystal cell development in flies. This brings strong support to the conclusion that the cell-based assay is suitable to identify genuine modulators of Srp/Lz activity and, more generally, of GATA/RUNX factors. However, further work will be required to discriminate between factors affecting GATA/RUNX interplay specifically or impinging also on either GATA or RUNX activity. Along this line, the results suggest that the three MED core modules but not the CDK8 regulatory module participate in Srp-induced transactivation. Importantly, too, 117 (90%) of the genes identified in the screen have well-conserved human homologs, suggesting they may regulate GATA/RUNX activity in humans. Actually, this sharp bias toward conserved genes underscores the fact that cell-based assays in Drosophila can serve as a powerful system to identify and characterize genes that may play similar roles in humans. Moreover, some homologs of Srp/Lz modifiers that were identified have been implicated in human diseases. These notably include MLF1, which is translocated in t(3;5)(q25.1;q34)-associated AML and whose Drosophila homolog is a target of Srp/Lz expressed in the crystal cells, as well as DDX10, which is translocated in inv(11)(p15q22)-associated AML. Whether these genes participate in GATA and/or RUNX function in normal or pathological situations in humans remains to be determined (Gobert, 2010).

The data show that the Mediator complex plays a central role in Srp/Lz-induced transactivation. Studies of yeast and metazoa highlighted the critical role of Mediator in both transcriptional activation and repression and showed that different Mediator subunits are required for the regulation of specific sets of genes or developmental processes. In addition, different transcription factors interact directly with specific Mediator subunits. Hence, the prevailing view is that different transcription factors depend on particular target proteins of the Mediator complex to regulate transcription. However, this study found that 20 of the 30 Mediator subunits were implicated as positive coregulators of Srp/Lz. Although some Mediator subunits, notably in the head module, play a global role in transcription, a general defect in transcription is unlikely to account for the observed decrease in Srp/Lz activity under the RNAi conditions, since no significant changes were observed in srp and Lz expression levels, except with Med19, a component of the head module, whose depletion decreased Lz levels. It is thus proposed that the integration of Srp/Lz transcriptional output requires the coordinated action of the different Mediator modules. However, it cannot be exclude that some of the Mediator subunits that were not identified in the screen may actually be dispensable for Srp/Lz activity (Gobert, 2010).

Remarkably, the CDK8 module, which is generally considered an accessory repressor module, was also required as a coactivator of Srp/Lz. Furthermore, in line with recent results revealing that all the functions of the CDK8 module do not rely on the CycC/Cdk8 pair, strong evidence is provided that only Med12/Med13 are required for the activation of Srp/Lz target genes in cell culture and in vivo. While different molecular mechanisms of repression by Cdk8/CycC and Med12/Med13 have been described, how Med12/Med13 may promote transcription remains elusive. These subunits may serve as an anchor to recruit the Mediator complex, as they have been shown to bind to Pygopus or ß-catenin to promote Wnt signaling and to Gli3 to inhibit Shh signaling. Accordingly, it was found that Srp and Lz interact with Med12 and Med13. However, this interaction could be due to another Mediator subunit required for Srp/Lz-induced transactivation. Alternatively, Med12/Med13 may be required for the proper folding of the Mediator complex to promote its interaction either with Srp/Lz or with downstream components of the transcriptional initiation machinery (Gobert, 2010).

In vivo, analysis of CDK8 module subunits shows that, reminiscent of what has been observed in larval imaginal discs, CycC/Cdk8 and Med12/Med13 have both common and specific functions during crystal cell development. Indeed, in the embryo, mutations in any of the four CDK8 module components resulted in a similar reduction in the absolute number of Lz+ blood cells and, concomitantly, of differentiated crystal cells, indicating that the whole CDK8 module controls the emergence of the crystal cell lineage. While the signaling that induces lz expression in the prohemocytes remains unknown, it was shown that the transcription factor Glial cell missing (Gcm) and the Friend of GATA corepressor U-shaped (Ush) oppose crystal cell fate choice. Both factors are expressed in the prohemocytes and interfere with lz expression to limit the number of crystal cells. Thus, loss of CDK8 module activity may impair crystal cell lineage emergence either by decreasing lz induction or by enhancing gcm or ush function (Gobert, 2010).

Similarly, it was found that targeted downregulation of Med12, CycC, or Cdk8 in the crystal cell lineage by RNAi after the onset of lz expression induced a cell-autonomous decrease in the absolute number of Lz+ larval blood cells. Hence, it is likely that the whole CDK8 module also controls the maintenance or the proliferation of the Lz+ cells during larval life. Recently, Wg signaling was shown to promote Lz+ larval blood cell proliferation. Interestingly, the CDK8 module participates in Wnt signaling. However, its coactivating function seemed to rely only on Med12/Med13 in Drosophila, whereas it depended on Cdk8/CycC in humans. Whether the CDK8 module regulates Lz+ cell number in response to Wg signaling or to another unknown pathway remains to be determined (Gobert, 2010).

In addition, the observation that only Med12 or Med13 downregulation caused a drop in the proportion of differentiated Lz+ cells in the larva strongly suggest that Med12/Med13 participates in crystal cell differentiation independently of the CycC/Cdk8 pair. In light of the results in cell culture, the in vivo data support an essential and direct function for Med12 and Med13 in the activation of the crystal cell differentiation program by Srp/Lz independently of CycC and Cdk8. All together, these data underline the functional flexibility of the CDK8 module, which appears to be reiteratively and specifically used at different stages of crystal cell development (Gobert, 2010).

In conclusion, it is anticipated that the results presented in this study lay the foundation for future investigations aiming at understanding the different levels of regulation of GATA and RUNX transcription factor activity not only in Drosophila but also in other species (Gobert, 2010).

A Smad action turnover switch operated by WW domain readers of a phosphoserine code

When directed to the nucleus by TGF-β or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. This study demonstrates that there is an order of events-Smad activation first and destruction later-and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-β pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers (see A Smad action turnover switch operated by WW domain readers of a phosphoserine code) provide an efficient solution to the problem of coupling TGF-β signal delivery to turnover of the Smad signal transducers (Aragón, 2011).

Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover

Notch signaling releases the Notch receptor intracellular domain (ICD), which complexes with CBF1 and Mastermind (MAM) to activate responsive genes. It has previously been reported that MAM interacts with CBP/p300 and promotes hyperphosphorylation and degradation of the Notch ICD in vivo. This study shows, in cultured HeLa cells, that CycC:CDK8 and CycT1:CDK9/P-TEFb are recruited with Notch and associated coactivators (MAM, SKIP) to the HES1 promoter in signaling cells. MAM interacts directly with CDK8 and can cause it to localize to subnuclear foci. Purified recombinant CycC:CDK8 phosphorylates the Notch ICD within the TAD and PEST domains, and expression of CycC:CDK8 strongly enhances Notch ICD hyperphosphorylation and PEST-dependent degradation by the Fbw7/Sel10 ubiquitin ligase in vivo. Point mutations affecting conserved Ser residues within the ICD PEST motif prevent hyperphosphorylation by CycC:CDK8 and stabilize the ICD in vivo. These findings suggest a role for MAM and CycC:CDK8 in the turnover of the Notch enhancer complex at target genes (Fryer, 2004).


Search PubMed for articles about Drosophila Cdk8

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Chang, Y. W., Howard, S. C. and Herman, P. K. (2004). The Ras/PKA signaling pathway directly targets the Srb9 protein, a component of the general RNA polymerase II transcription apparatus. Mol. Cell 15: 107-116. PubMed ID: 15225552

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Chung, H. L., Mao, X., Wang, H., Park, Y. J., Marcogliese, P. C., Rosenfeld, J. A., Burrage, L. C., Liu, P., Murdock, D. R., Yamamoto, S., Wangler, M. F., Chao, H. T., Long, H., Feng, L., Bacino, C. A., Bellen, H. J. and Xiao, B. (2020). De Novo Variants in CDK19 Are Associated with a Syndrome Involving Intellectual Disability and Epileptic Encephalopathy. Am J Hum Genet. PubMed ID: 32330417

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Gobert, V., et al. (2010). A genome-wide RNA interference screen identifies a differential role of the mediator CDK8 module subunits for GATA/ RUNX-activated transcription in Drosophila. Mol. Cell Biol. 30(11): 2837-48. PubMed ID: 20368357

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date revised: 10 December 2020

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