#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

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


Authors: Xiao Li aff001;  Mengmeng Liu aff001;  Xingjie Ren aff002;  Nicolas Loncle aff003;  Qun Wang aff001;  Rajitha-Udakara-Sampath Hemba-Waduge aff001;  Stephen H. Yu aff001;  Muriel Boube aff003;  Henri-Marc G. Bourbon aff003;  Jian-Quan Ni aff002;  Jun-Yuan Ji aff001
Authors place of work: Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M University Health Science Center, Bryan, Texas, United States of America aff001;  School of Medicine, Tsinghua University, Beijing, China aff002;  Centre de Biologie Intégrative, Centre de Biologie du Développement, UMR5544 du CNRS, Université de Toulouse, Toulouse, France aff003;  Department of Nutrition, Texas A&M University, College Station, Texas, United States of America aff004
Published in the journal: The Mediator CDK8-Cyclin C complex modulates Dpp signaling in Drosophila by stimulating Mad-dependent transcription. PLoS Genet 16(5): e32767. doi:10.1371/journal.pgen.1008832
Category: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008832

Summary

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. Thus, it is essential to understand the regulatory network modulating the CDK8-CycC complex in both normal development and tumorigenesis. To identify upstream regulators or downstream effectors of CDK8, we performed a dominant modifier genetic screen in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. We identified 26 genomic loci whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led us to identify 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. We 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, our 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.

Keywords:

Gene expression – Drosophila melanogaster – Gene regulation – Phenotypes – Phosphorylation – Transcriptional control – Suppressor genes – DPP signaling cascade

Introduction

Composed of up to 30 conserved subunits, the Mediator complex plays critical roles in modulating RNA polymerase II (Pol II)-dependent gene expression by functioning as a molecular bridge linking transcriptional activators and the general transcription machinery in almost all eukaryotes [15]. Biochemical purification of the human Mediator complex has revealed the Cyclin-Dependent Kinase 8 (CDK8) module, composed of CDK8 (or its paralogue CDK19, also known as CDK8L), CycC, Med12 (or Med12L), and Med13 (or Med13L), and the small Mediator complex, composed of 26 subunits that are divided into the head, middle, and tail modules [69]. CDK8 is the only Mediator subunit with enzymatic activities. The CDK8 kinase module (CKM) has been proposed to function in two modes. First, it can reversibly bind with the small Mediator complex to form the large Mediator complex, thereby physically blocking the interaction between the small Mediator complex and the general transcription machinery (notably with RNA Pol II itself). Second, CDK8 can function as a kinase that phosphorylates different substrates, particularly transcriptional activators such as E2F1 [10,11], N-ICD (intracellular domain of Notch) [12], p53 [13], Smad proteins [14,15], SREBP (sterol regulatory element-binding protein) [16], and STAT1 (signal transducer and activator of transcription 1) [17]. These characterized functions of CDK8 highlight fundamental roles of the CKM in regulating transcription.

Besides its roles in specific developmental and physiological contexts, the CKM subunits are dysregulated in a variety of human diseases, such as cancers [1822]. For example, CDK8 has been reported to act as an oncoprotein in melanoma and colorectal cancers [10,23,24]. Moreover, CDK8 and CDK19 are overexpressed in invasive ductal carcinomas, correlating with shorter relapse-free survival in breast cancer [25]. Gain or amplification of CDK8 activity is sufficient in driving tumorigenesis in colorectal and pancreatic cancers in human, as well as in skin cancer in fish [14,23,2628]. Because of these discoveries, there is a considerable interest in developing drugs targeting the CDK8 kinase for cancer treatment in recent years [29,30]. However, exactly how CDK8 dysregulation contributes to tumorigenesis remains poorly understood. Thus it is essential to reveal the function and regulation of CDK8 activity in different developmental, physiological, and pathological processes.

The major bottleneck for addressing these critical gaps in our knowledge is the lack of in vivo readouts for CDK8-specific activities in metazoans. We overcame this challenge by generating tissue-specific phenotypes caused by varying CDK8 activities in Drosophila. After validating the specificity of these phenotypes using genetic, molecular, and cell biological approaches, we performed a dominant modifier genetic screen to identify factors that interact with CDK8 in vivo based on these unique readouts for CDK8-specific activities. From the screen, we identified Dad (Daughters against dpp), which encodes an inhibitory Smad in the Dpp (Decapentaplegic)/TGFβ (Transforming Growth Factor-β) signaling pathway, as well as additional components of the Dpp signaling pathway including dpp, tkv (thickveins, encoding the type I receptor for Dpp), Mad (Mothers against dpp) and Medea (encoding the Smad1/5 and Smad4 homologs, respectively) in Drosophila. Consistent with the previous biochemical analyses suggesting that CDK8 may phosphorylate Drosophila Mad or human Smad1 [14,15,31], thereby regulating their transcriptional activities [14,15,31], our results have validated and further advanced our understanding of this conserved regulatory mechanism in vivo. Furthermore, our analyses have revealed additional Mediator subunits and protein kinases involved in regulating the Mad/Smad-dependent transcription. These results, together with previous studies, suggest that concerted recruitment of the Mediator complexes and other cofactors play a pivotal role in regulating Mad/Smad-dependent gene expression, a critical process for TGFβ signaling to function in a variety of biological and pathological contexts.

Results

Wing vein patterning defects caused by varying the levels of CDK8, CycC, or both

To study the function and regulation of CDK8 and CycC in vivo, we have generated transgenic lines to either deplete them by RNAi (RNA interference) or conditionally overexpress the wild-type CDK8 kinase using the Gal4-UAS system [32,33]. Normal Drosophila wings display stereotypical vein patterns, consisting of six longitudinal veins, dubbed L1 to L6, and two crossveins, the anterior crossvein and the posterior crossvein (Fig 1A). Knocking down of CDK8 using the nub-Gal4 (nubbin-Gal4) line (see Materials and Methods for details), which is specifically expressed in the wing pouch area of the wing imaginal discs [34], results in the formation of ectopic veins in the intervein region, especially around L2 and L5 (Fig 1B). Similar phenotypes were observed with the depletion of CycC (Fig 1C) or both CDK8 and CycC (Fig 1D). In contrast, overexpression of wild-type CDK8 (UAS-Cdk8+) disrupts the L3 vein, the L4 vein, and the crossveins (Fig 1E), opposite to the phenotypes caused by depleting CDK8, CycC, or both. However, overexpression of a kinase-dead (KD) form of CDK8 (UAS-Cdk8KD) using the same approach does not affect the vein patterns (Fig 1F), suggesting that the effects of CDK8 on vein phenotypes are dependent on the kinase activity of CDK8. These observations show that CDK8 and CycC are involved in regulating the vein patterning in Drosophila.

Wing vein patterning defects caused by varying the levels of CDK8, CycC, or both.
Fig. 1. Wing vein patterning defects caused by varying the levels of CDK8, CycC, or both.
Adult female wings of (A) nub-Gal4/+ (control), note the longitudinal veins L1-L6, anterior crossvein (ACV), and posterior crossvein (PCV); (B) w1118/+; nub-Gal4/+; UAS-Cdk8-RNAi/+; (C) w1118/+; nub-Gal4/+; UAS-CycC-RNAi/+; (D) w1118/+; nub-Gal4/+; UAS-Cdk8-RNAi CycC-RNAi/+; (E) w1118/+; nub-Gal4>UAS-Cdk8+/+; (F) w1118/+; nub-Gal4/UAS-Cdk8KD; (G) w1118/+; nub-Gal4>UAS-Cdk8+/+; cdk8K185; and (H) w1118/+; nub-Gal4/+; UAS-Cdk8-RNAi/cdk8K185.

Interestingly, depletion of CDK8 (Fig 1B), CycC (Fig 1C), or both (Fig 1D) increase the size of the wings, correlating to a significant increase of total cell numbers but a reduction of cell sizes (S1 Fig). In contrast, overexpression of wild-type CDK8 reduces the size of wings and total cell numbers, but no obvious effects on cell size (Fig 1E and S1 Fig). The effects of CDK8 on wing size can also be visualized using ap-Gal4 (apterous-Gal4), which is specifically expressed within the dorsal compartment of the wing discs (Fig 2A) [35]. Ap-Gal4-induced depletion of CDK8 and CycC caused the adult wing to curl downwards (S2C Fig), indicating the overgrowth of the dorsal compartment compared to the ventral compartment; while overexpression of CDK8 led to the adult wing to curl upwards (S2D Fig), suggesting reduced growth of the dorsal compartment. We have previously reported that CDK8 inhibits the transcriptional activity of E2F1, a key transcription factor that controls the expression of factors required for the G1 to S-phase transition of the cell cycle [10,11]. Thus, the effects of CDK8 levels on wing size and cell numbers are likely through E2F1-regulated cell-cycle progression.

Validation of the specificity of the vein defects caused by depletion or overexpression of CDK8-CycC.
Fig. 2. Validation of the specificity of the vein defects caused by depletion or overexpression of CDK8-CycC.
Representative confocal images of the wing pouch area of a L3 wandering larval wing disc: (A) ap-Gal4/UAS-2XGFP with DAPI (blue) and GFP (green); (B) ap-Gal4/+ with anti-CDK8 (red) staining; (C) ap-Gal4/+ with anti-CycC (red) staining; (D) ap-Gal4/+; UAS-Cdk8-RNAi/+ with anti-CDK8 (red) staining; (E) ap-Gal4/+; UAS-CycC-RNAi/+ with anti-CycC (red) staining; (F) ap-Gal4/+; UAS-Cdk8-RNAi CycC-RNAi/+ with anti-CDK8 (red) staining; (G) ap-Gal4/+; UAS-Cdk8-RNAi CycC-RNAi/+ with anti-CycC (red) staining; (H) ap-Gal4/UAS-Cdk8+ with anti-CDK8 (red) staining; and (I) ap-Gal4/UAS-Cdk8KD with anti-CDK8 (red) staining. Note that the gain for confocal imaging in H and I is lower than the others to avoid over saturation of the signals. At least five wing discs were examined for each genotype. The dorsal/ventral (D/V) boundary is shown in A, D and H. Scale bar in I: 25μm.

Validation of the specificity of the vein defects caused by depletion or overexpression of CDK8-CycC

To verify the specificity of these phenotypes, we recombined the nub-Gal4 line with the CDK8-RNAi, CycC-RNAi, or CDK8-overexpression lines, and then tested whether these vein phenotypes could be dominantly modified by cdk8K185, a null allele of cdk8 [36]. As shown in Fig 1G, reducing CDK8 by half in a ‘cdk8K185/+’ heterozygous background suppresses the vein defects caused by CDK8 overexpression. However, heterozygosity of cdk8K185 does not obviously enhance the vein phenotype caused by CDK8-RNAi (Fig 1H), indicating that the RNAi of CDK8 may have depleted most of the CDK8 protein pool.

To further validate the specificity of the CDK8-directed phenotypes at the cellular level, we analyzed the protein levels of CDK8 and CycC in wing discs at the third instar wandering larval stage by immunostaining with CDK8 or CycC specific antibodies. Normally, both the CDK8 (Fig 2B) and CycC (Fig 2C) proteins are uniformly distributed in the nuclei of all wing disc cells. Depletion of CDK8 (Fig 2D), CycC (Fig 2E), or both (Fig 2F and 2G) using the ap-Gal4 line significantly reduced CDK8 or CycC proteins in the dorsal compartment. The ventral compartment of the same discs serves as the internal control. In contrast, overexpression of either wild-type (Fig 2H) or kinase-dead (Fig 2I) CDK8 using ap-Gal4 specifically increased the levels of CDK8 protein in the dorsal compartment. Taken together, these genetic and cell biological analyses have validated the specificity of both the antibodies and transgenic lines, demonstrating that these vein phenotypes are caused by a specific gain or reduction of CDK8 activity in vivo.

Identification of deficiency lines that can dominantly modify the vein phenotypes caused by varying CDK8

Based on these CDK8-specific vein phenotypes, we performed a dominant modifier genetic screen to identify gene products that can functionally interact with CDK8 in vivo. The idea of using phenotypic modifications to identify multiple genes involved in determining a specific trait or a phenotypic endpoint was initially developed by Calvin B. Bridges, when he analyzed mutant genes that could interact with the eosin mutant in regulating eye color in flies [37]. This genetic modifier approach has been employed to reveal the functional and inter-molecular networks for proteins of interest in Drosophila (for instances, [3842]), and to provide insights into the phenotypic and genetic variability in mammals [43,44]. The approach posits that if a protein interacts with CDK8-CycC in vivo in defining the wing vein patterns, then reducing its level by half may either enhance or suppress the sensitized wing vein phenotypes caused by specific alteration of the CDK8 activities. Accordingly, we can survey through the fly genome to search for factors that interact with CDK8-CycC using single genetic crosses.

To facilitate this screen approach, we generated three stocks with the following genotypes: “w1118; nub-Gal4; UAS-Cdk8-RNAi” (designated as “nub>Cdk8-i” for simplicity), “w1118; nub-Gal4; UAS-CycC-RNAi” (“nub>CycC-i”), and “w1118; nub-Gal4, UAS-Cdk8+/CyO” (“nub>Cdk8+”). We then conducted a dominant modifier genetic screen by crossing these three lines in parallel with a collection of 490 deficiency (Df) lines (S1 Table), which uncovers the majority of the euchromatic genome [45,46]. Any alteration of the wing vein patterns can be readily discerned under dissecting microscopes, allowing us to search for Df lines that could modify the vein phenotypes caused by specific alteration of CDK8 activities.

We inspected the vein patterns of the F1 females for enhancers and suppressors based on the following criteria: suppressors of the CDK8- or CycC-RNAi phenotypes are expected to display fewer or no ectopic veins (e.g., Fig 3A and 3C), while enhancers of the CDK8- or CycC-RNAi phenotypes show more or longer ectopic veins (e.g., Fig 3B and 3D). To score the strength of the modifications, we define strong suppressors as the Df lines that eliminate all of the ectopic veins, while the Df lines that only shorten the length of the ectopic veins are scored as weak suppressors. Similarly, we define strong enhancers to cause more or longer ectopic veins than CDK8- or CycC-RNAi phenotypes, while the Df lines causing less severe vein defects are designated as the weak enhancers. Conversely, the strong suppressors of the CDK8-overexpression phenotype are expected to have vein patterns similar to those of wild-type wings (particularly the L3/L4; e.g., Fig 3E, compared to the control shown in Fig 1E). If the Df lines only partially restore the missing veins, then they are scored as the weak suppressors. In contrast, the strong enhancers of the CDK8-overexpression phenotype are defined by further disrupting the vein patterns, with the entire L3 or L4 missing, often accompanied with strong disruption on other veins (e.g., Fig 3F); while the weaker enhancers further disrupted the vein defects compared to the CDK8-overexpression phenotype, but less severe than the strong enhancers.

Identification of deficiency lines that can dominantly modify the vein phenotypes caused by varying CDK8.
Fig. 3. Identification of deficiency lines that can dominantly modify the vein phenotypes caused by varying CDK8.
(A-F) Adult wings showing the examples of dominant modifiers. (A) nub-Gal4/Df(2R)Exel6064; UAS-Cdk8-RNAi (a suppressor of the CDK8-RNAi phenotype); (B) nub-Gal4/+; UAS-Cdk8-RNAi/Df(3R)Exel6176, (an enhancer of the CDK8-RNAi phenotype); (C) nub-Gal4/Df(2R)Exel6064; UAS-CycC-RNAi/+ (a suppressor of the CycC-RNAi phenotype); (D) nub-Gal4/+; UAS-CycC-RNAi/Df(3R)Exel6176 (an enhancer of the CycC-RNAi phenotype); (E) nub-Gal4>UAS-Cdk8+/+; Df(3R)Exel6176 /+ (a suppressor of the CDK8-overexpression phenotype); and (F) nub-Gal4>UAS-Cdk8+/Df(2R)Exel6064 (an enhancer of the CDK8-overexpression phenotype). Scale bar in F: 0.4mm. (G and H) The Venn diagrams summarize the numbers of suppressors and enhancers of the CDK8-specific phenotypes.

From these screens, we identified 57 suppressor and 90 enhancer Df lines for the CDK8-RNAi phenotype, and 62 suppressor and 98 enhancer Df lines for the CycC-RNAi phenotype. In addition, we identified 63 enhancer and 98 suppressor Df lines for the CDK8-overexpression phenotype (Fig 3G and 3H). The results for all of these Df lines are summarized in S1 Table. Of these dominant modifier Df lines, four of them suppressed the CDK8-RNAi and CycC-RNAi phenotypes but enhance the CDK8-overexpression phenotype (Fig 3G, Table 1), while 22 of them enhance the CDK8-RNAi and CycC-RNAi phenotypes but suppress the CDK8-overexpression phenotype (Fig 3H, Table 1). To further validate this genetic approach, we generated a transgenic line that allowed us to simultaneously deplete CDK8 and CycC (“w1118; nub-Gal4; UAS-Cdk8-RNAi, CycC-RNAi”, referred to as “nub>Cdk8-i CycC-i”) with nub-Gal4, and observed identical phenotypes to the ones caused by depleting either Cdk8 or CycC alone (Fig 1D). With the exception of one Df line, the rest of these 25 Df lines have consistently modified the ectopic vein phenotype caused by depletion of both CDK8 and CycC: four of the Df lines behaved as suppressors and 21 of them as enhancers (Table 1). These results show that the CDK8-specific vein phenotypes are modifiable and can be used to identify factors that functionally interact with CDK8-CycC in vivo.

Tab. 1. Deficiency lines that dominantly modify the CDK8- or CycC-specific phenotypes.
Deficiency lines that dominantly modify the CDK8- or CycC-specific phenotypes.

Identification of Dad as an enhancer of the nub>Cdk8-i and nub>CycC-i phenotypes but a suppressor of the Cdk8-overexpression phenotype

To identify the specific genes uncovered by these dominant modifier Df lines, we analyzed these 26 genome regions with partial overlapping Df lines (Table 1). Interestingly, two partially overlapping Df lines, Df(3R)BSC748 and Df(3R)Exel6176, enhanced the CDK8-RNAi and CycC-RNAi phenotypes, but suppressed the CDK8-overexpression phenotype (Fig 3B, 3D and 3E; Table 1). The overlapping region uncovers one specific gene, Dad (Daughter against Dpp), encoding the Drosophila homolog of Smad6/7 (Fig 4A). Thus, to test whether Dad is the specific gene that accounts for the modification of the CDK8-specific phenotypes by these two Df lines, we performed similar genetic tests with two mutant alleles of Dad: DadMI04922, a MiMIC (Minos Mediated Integration Cassette) insertion in an intron of the Dad gene [47], and Dadj1E4, an insertion of the P{lacW} element in an intron of the Dad gene [48]. Indeed, both Dad mutant alleles dominantly enhanced the CDK8-RNAi (Fig 4B), CycC-RNAi (Fig 4C), and CDK8-RNAi plus CycC-RNAi (Fig 4D) phenotypes, but suppressed the CDK8-overexpression phenotype (Fig 4E, Table 2). These effects on the CDK8-specific vein phenotypes are similar to those observed for Df(3R)BSC748 and Df(3R)Exel6176, suggesting that Dad is the specific gene that genetically interacts with CDK8 in vivo.

Identification of the <i>Dad</i> gene and genes encoding other components of the Dpp signaling pathway as dominant modifiers of the CDK8-specific phenotypes.
Fig. 4. Identification of the Dad gene and genes encoding other components of the Dpp signaling pathway as dominant modifiers of the CDK8-specific phenotypes.
(A) Schematic diagram of the genome region of Df(3R)BSC748 and Df(3R)Exel6176, which uncover the gene dad. Adult wings with the following genotypes: (B) nub-Gal4/+; UAS-Cdk8-RNAi/DadMI04922; (C) nub-Gal4/+; UAS-CycC-RNAi/DadMI04922; (D) nub-Gal4/+; UAS-Cdk8-RNAi CycC-RNAi/DadMI04922; (E) nub-Gal4>UAS-Cdk8+/+; DadMI04922/+; (F) nub-Gal4/tkvk16713; UAS-Cdk8-RNAi/+; (G) nub-Gal4>UAS-Cdk8+/tkvk16713; (H) nub-Gal4/Mad12; UAS-Cdk8-RNAi/+; (I) nub-Gal4>UAS-Cdk8+/Mad12; (J) nub-Gal4/+; UAS-Cdk8-RNAi/Medea13; and (K) nub-Gal4>UAS-Cdk8+/+; Medea13/+; Scale bar in K: 0.4mm.
Tab. 2. Mutant alleles of genes encoding components of the Dpp signaling that modify the CDK8- or CycC-specific phenotypes.
Mutant alleles of genes encoding components of the Dpp signaling that modify the CDK8- or CycC-specific phenotypes.

Mutants of multiple components of the Dpp signaling pathway genetically interact with CDK8-CycC

The protein Dad functions as an inhibitory Smad in the Dpp/TGFβ signaling pathway, which plays critical roles in regulating cell proliferation and differentiation during the development of metazoans [4954]. During the development of the wing discs, Dpp spreads from the anterior-posterior boundary to the anterior and posterior halves [4951,55]. Upon the binding of the Dpp ligand to the Tkv-Punt receptor complex on the cell membrane, the TGFβ type II receptor Punt phosphorylates and activates the type I receptor Tkv. This results in the phosphorylation of Mad by Tkv at its C-terminal SSXS motif, known as the phospho-Mad protein or pMad. Medea, the unique co-Smad protein in Drosophila, associates with pMad in the cytoplasm, and then this heteromeric Smad complex translocates into the nucleus and regulates the expression of its target genes [53,5557].

The genetic interactions between CDK8-CycC and Dad prompted us to test whether mutant alleles of other components of the Dpp signaling pathway could also genetically interact with CDK8 and CycC. For this, we crossed multiple mutant alleles of these components with the CDK8-CycC depletion or overexpression lines. As summarized in Table 2, mutants of multiple components of the Dpp signaling pathway could either dominantly enhance or suppress the CDK8-specific vein phenotypes. For instance, dppd6, dpphr92, dppS11, tkv7, tkvk16713 (Fig 4F), Mad1-2, Mad12 (Fig 4H), Mad8-2, Madk00237, MadKG00581, Medea1, and Medea13 (Fig 4J) all dominantly suppress the ectopic vein phenotype caused by depletion of CDK8, CycC, or both CDK8 and CycC (Table 2). However, tkv7, tkvk16713 (Fig 4G), Madk00237, Mad12 (Fig 4I), MadKG00581, Medea1, and Medea13 (Fig 4K) enhance the CDK8-overexpression phenotype (Table 2). Testing additional mutant alleles of these genes have revealed that most of them can also dominantly modify the CDK8-specific phenotypes (Table 2). Dpp is activated in a specific pattern in the middle part of the wing pouch area, while the nub-Gal4 display a well-characterized pattern in the entire wing pouch area. These two patterns differ, arguing against the possibility that Dpp signaling may affect nub-Gal4 expression pattern. In addition, reducing Mad or Dad by half has little effects on the expression of a UAS-RFP reporter driven by nub-Gal4 (S3 Fig), suggesting that the expression and activity of nub-Gal4 are not affected by Dpp signaling. Taken together, these genetic interactions suggest that CDK8-CycC may affect vein patterning by modulating Dpp signaling.

CDK8-CycC positively regulates Mad-dependent transcription

Given that CDK8 and CycC are known subunits of the Mediator complex, which serves as a scaffold complex mediating the interactions between the RNA Pol II basal transcription machinery and a number of gene-specific transcription activators [3,7,58]. Thus, the simplest model to explain the genetic interactions between Dpp signaling and CDK8-CycC is that the CDK8-CycC complex may directly regulate the transcriptional activity of Mad in the nucleus. To test this model, we analyzed the effects of CDK8-CycC depletion on the expression of salm (spalt major), a well-characterized direct target gene of Mad involved in vein differentiation [5962]. The sal-lacZ (P{PZ}salm03602) is a enhancer trap line derived from an insertion of a P{PZ} element in the promoter region of the salm gene [63,64], and the expression of sal-lacZ can serve as a reporter for the transcriptional activity of Mad [65].

Because the expression of sal-lacZ is symmetric along the dorsal-ventral boundary of the wing pouch area of the wing discs (Fig 5A), we tested whether specific depletion of CDK8 or CycC within the dorsal compartment of the wing discs could affect the transcriptional activity of Mad by detecting the transcription level of sal using an anti-β-galactosidase (anti-β-Gal) antibody. For this, we depleted genes of interest using the ap-Gal4 driver, and then compared the β-Gal expression between the dorsal and ventral compartments. As expected, depleting Mad with two transgenic RNAi lines (BL-43183 (Fig 5B) and BL-31315 (S4B and S4B’ Fig)), Medea (Fig 5C), or Dpp (S4A Fig) using this approach reduced the expression of the sal-lacZ reporter in the dorsal compartment. Importantly, depletion of CDK8 (Fig 5D), CycC (Fig 5E), or both (Fig 5F), in the dorsal compartment significantly decreased the β-Gal expression level in the dorsal compartment compared with the ventral compartment of the same disc. After quantifying the line-scan profiles of the Sal-lacZ levels in the wing porch area, we calculated the relative signal intensity of dorsal to ventral Sal-lacZ levels for 5 wing discs of each genotype (S5 Fig; see Materials and Methods for details), which validated the effects of CDK8-CycC on sal-lacZ expression (Fig 5G). Similar observations were made by quantification of the pixel intensities in areas in the dorsal and ventral compartments (S6 Fig).

CDK8-CycC positively regulates Mad-dependent transcription.
Fig. 5. CDK8-CycC positively regulates Mad-dependent transcription.
Confocal images of the wing pouch area of a L3 wandering larval wing disc of (A) ap-Gal4, sal-lacZ/+ (control); (B) ap-Gal4, sal-lacZ/UAS-Mad-RNAi (BL-43183); (C) ap-Gal4, sal-lacZ/UAS-Medea-RNAi; (D) ap-Gal4, sal-lacZ/+; UAS-Cdk8-RNAi/+; (E) ap-Gal4, sal-lacZ/+; UAS-CycC-RNAi/+; and (F) ap-Gal4, sal-lacZ/+; UAS-Cdk8-RNAi CycC-RNAi/+. All signals presented were from anti-β-galactosidase staining. Scale bar in F: 25μm. Dorsal (D)-ventral (V) boundaries are marked using a short line in these images. (G) Quantification of Sal-lacZ expression. The black columns represent the average of Sal-lacZ expression in the ventral compartment of the indicated genotypes (N = 5 for each genotype), and light green columns represent the measurements in the dorsal compartments. (H) Western Blots of a GST pull-down assay between GST-CDK8 and His-tagged Mad fragments. The amino acids (AA) positions of MH1 and MH2, separated by the linker region, are based on a BLAST search of Drosophila Mad-RA isoform (455AA). The other isoform, Mad-RB (525AA), has additional 70AA at the N-terminus. We focused on the Mad-RA isoform in this work. (I) Y2H assay showing the specific interaction between CDK8 and the linker region of Mad. SD/-Leu/-Trp and SD/-Leu/-Trp/-His are synthetic dropout (SD) media lacking “Leu and Trp”, or “Leu, Trp, and His”, respectively. The co-transformed yeast cultures were spotted on SD/-Leu/-Trp and SD/-Leu/-Trp/-His plates, positive interactions result in yeast growth on the SD/-Leu/-Trp/-His plate. AD, GAL4-activation domain (prey); BD, GAL4-DNA-binding domain (bait); AD- or BD-protein, AD- or BD-fusion proteins.

To further validate the effects of CDK8-CycC depletion on Mad-activated gene expression, we analyzed the expression of the quadrant enhancer (QE) of the selector gene vestigial (vgQE-lacZ) in wing discs. Similar to Sal-lacZ reporter, vgQE-lacZ also displays a symmetric expression pattern along the D-V boundary in the wing pouch (Fig 6A) [66,67]. As expected, depleting Mad (BL-31315) using ap-Gal4 driver significantly reduced the expression of vgQE-lacZ in the dorsal compartment (Fig 6B). Although depleting CDK8 alone only marginally reduced the vgQE-lacZ expression in the dorsal compartment (Fig 6C), a more obvious effect was observed with the depletion of CycC (Fig 6D), and a stronger reduction of the reporter expression was detected with the depletion of both CDK8 and CycC (Fig 6E) using the same approach. We note that the interpretation of the data presented in Fig 6 is compounded by the fact that the transcription of the vg in different compartments of wing discs is controlled by Wingless (Wg) and Dpp signaling, as well as a feed-forward regulation by Vg itself [66,67]. Nevertheless, the most parsimonious model to explain the observations based on Sal-lacZ and vgQE-lacZ reporters is that CDK8-CycC positively regulates Mad-dependent transcription.

Effects of various Mediator subunits on the expression of the <i>vgQE-lacZ</i> reporter.
Fig. 6. Effects of various Mediator subunits on the expression of the vgQE-lacZ reporter.
Representative confocal images of anti-β-Gal staining of wing discs of the following genotypes: (A) ap-Gal4/+; vgQE-lacZ/+; (B) ap-Gal4/+; vgQE-lacZ/UAS-Mad-RNAi (BL-31315); (C) ap-Gal4/+; vgQE-lacZ/UAS-Cdk8-RNAi; (D) ap-Gal4/+; vgQE-lacZ/UAS-CycC-RNAi; (E) ap-Gal4/+; vgQE-lacZ/UAS-Cdk8-RNAi CycC-RNAi; (F) ap-Gal4/+; vgQE-lacZ/UAS-Med12-RNAi; (G) ap-Gal4/+; vgQE-lacZ/UAS-Med13-RNAi; (H) ap-Gal4/+; vgQE-lacZ/UAS-Med15-RNAi; (I) ap-Gal4/+; vgQE-lacZ/UAS-Med23-RNAi; (J) ap-Gal4/+; vgQE-lacZ/UAS-Med24-RNAi; (K) ap-Gal4/+; vgQE-lacZ/UAS-Med31-RNAi; (L) ap-Gal4/+; vgQE-lacZ/UAS-Med30-RNAi. At least five wing discs were examined for each genotype. Scale bar in L: 25μm.

One caveat of these analyses is that the CKM could affect ap-Gal4 activities. As shown in S7B Fig, we observed that depleting CDK8 and CycC reduces the ap-Gal4-dependent expression of UAS-GFP in the dorsal compartment of wing discs (compared to the control shown in S7A Fig). This observation suggests that the positive effects of depletion of CDK8 and CycC on wing vein patterning are hypomorphic, representing an under-estimation of the positive effects of CDK8-CycC in regulating Mad-dependent transcription. In addition, we observed that depleting Ap protein using ap-Gal4 has no effects on the sal-lacZ expression in the dorsal compartment (S7C Fig), suggesting that the expression of sal-lacZ is independent of the levels of Ap or Gal4.

Direct interactions between CDK8 and Mad

Since Mad phosphorylation at its C-terminus (pMad) by the Tkv-Punt receptor complex marks the activation of Mad, we tested whether CDK8 affects the pMad level. For this, we depleted CDK8, CycC, or both, with the ap-Gal4 line, and then detected the levels of the activated Mad with an anti-pMad antibody. In the wing pouch area of the control discs, the pMad protein is symmetrically distributed along the dorsal-ventral boundary (S8A Fig). However, depletion of CDK8-CycC did not affect pMad levels when comparing the dorsal compartment with the ventral compartment (S8B–S8D Fig), suggesting that CDK8-CycC does not affect the phosphorylation of Mad at its carboxy terminus in the cytoplasm. These results support the idea that the CDK8-CycC complex directly regulates the transcriptional activity of Mad in the nucleus.

R-Smads are characterized by a highly conserved amino-terminal MH1 (Mad homology 1) domain that binds to DNA and a C-terminal MH2 (Mad homology 2) domain that harbors the transactivation activity, separated by a serine- and proline-rich linker region (Fig 5H) [68]. It was previously reported that CDK8 and a few other kinases (see below) may phosphorylate Smad proteins in both Drosophila and mammalian cells [14,15,31,55,68], but whether and how CDK8 interacts with Smads remain unknown. To determine whether CDK8 directly interacts with Mad, we performed a GST-pulldown assay. As shown in Fig 5H, purified GST-CDK8 can directly bind with His-tagged full length Mad (Mad-FL, AA1-455) expressed in E. coli. We then mapped the specific domain of Mad that interacts with CDK8 using His-tagged fragments of the Mad protein. We observed that the “Mad-N2” fragment (AA1-230) and the “Mad-C2” fragment (AA151-455), but not the “Mad-N1” fragment (AA1-150) or the “Mad-C1” fragment (AA231-455), can interact directly with CDK8 (Fig 5H). We validated the interaction between CDK8 and the linker region using the yeast two-hybrid (Y2H) assay: the “Mad-N2” fragment, but not the “Mad-N1” fragment, as the bait can interact with full-length CDK8 as the prey (Fig 5I). It is not feasible to use this Y2H approach test with Mad-FL or Mad-C1/C2 fragments as bait, since they auto-activate as the baits; while the full-length CDK8 can also auto-activate as the bait (S9 Fig). Taken together, these results suggest that CDK8 directly interacts with part of the linker region of Mad protein (AA151-230). Implications of these physical interactions are discussed below.

Involvement of additional Mediator complex subunits in regulating the Mad/Smad-dependent transcription

The Med15/ARC105 subunit of the Mediator complex has been previously shown to directly interact with the transactivation MH2 domain of Smad2/3, thereby mediating the Smad2/3-Smad4-dependent transcription in Xenopus [69], and Med15 is required for the transcription of Dpp target genes in Drosophila [70]. However, whether other Mediator subunits are involved in regulating the Mad/Smad-dependent transcription remains unknown. To address this question, we depleted individual subunits of the Mediator complex upon conditional expression of interfering RNAs with ap-Gal4, and then analyzed the expression of the sal-lacZ reporter. Of the 30 Mediator subunits tested (Table 3), we have observed that depletion of six additional Mediator subunits, Med12 (Fig 7B), Med13 (Fig 7C), Med15 (Fig 7D), Med23 (Fig 7E), Med24 (Fig 7F), and Med31 (Fig 7G), by ap-Gal4 significantly reduced the expression of sal-lacZ in cells of the dorsal compartment compared with the cells in the ventral compartment of the same wing discs (Fig 7J); similar to depletion of CDK8 or CycC (Fig 5). The effects of these six Mediator subunits were further validated using the vgQE-lacZ reporter: their depletion using ap-Gal4 also reduces the vgQE-lacZ expression in the dorsal compartment (Fig 6F–6K). These results suggest that these Mediator subunits are required for the Mad-activated gene expression. However, RNAi depletion of the remaining 15 Mediator subunits using ap-Gal4 driver did not significantly affect sal-lacZ expression (Table 3), as β-Gal expression remained symmetric along the dorsal-ventral boundary as exemplified for depletion of Med1 (Fig 7A and 7J) and Med25 (Fig 7H) on sal-lacZ expression. Similarly, depletion of Med30 using ap-Gal4 does not obviously affect the expression of sal-lacZ and vgQE-lacZ reporters, which remains symmetric along the dorsal-ventral boundary (Fig 6L, Table 3). Furthermore, depleting the remaining Mediator subunits, including Med7 (Fig 7I), Med8 (S10A Fig), Med14 (S10B Fig), Med16 (S10C Fig), Med17 (S10D Fig), Med21 (S10E Fig), and Med22 (S10F Fig), severely disrupted the morphology of the wing discs, making it difficult to determine their roles in regulating sal transcription. Taken together, these observations suggest that multiple Mediator subunits, but apparently not all of them, are required for Mad-dependent transcription in Drosophila.

Effects of the additional Mediator subunits on the expression of the <i>sal-lacZ</i> reporter.
Fig. 7. Effects of the additional Mediator subunits on the expression of the sal-lacZ reporter.
Representative confocal images of anti-β-Gal staining of wing discs of the following genotypes: (A) ap-Gal4, sal-lacZ/+; UAS-Med1-RNAi/+; (B) ap-Gal4, sal-lacZ/+; UAS-Med12-RNAi/+; (C) ap-Gal4, sal-lacZ/+; UAS-Med13-RNAi/+; (D) ap-Gal4, sal-lacZ/+; UAS-Med15-RNAi/+; (E) ap-Gal4, sal-lacZ/+; UAS-Med23-RNAi/+; (F) ap-Gal4, sal-lacZ/+; UAS-Med24-RNAi/+; (G) in ap-Gal4, sal-lacZ/+; UAS-Med31-RNAi/+; (H) ap-Gal4, sal-lacZ/UAS-Med25-RNAi; and (I) ap-Gal4, sal-lacZ/+; UAS-Med7-RNAi/+. (J) Quantification of Sal-lacZ expression. The black columns represent the average of Sal-lacZ expression in the ventral compartment of five wing discs of the indicated genotypes (N = 5 for each genotype), and light green columns represent the measurements in the dorsal compartments. Scale bar in A: 25μm. For (H) and (I), at least five wing discs were examined for each genotype.
Tab. 3. The effects of depleting different Mediator subunits on the expression of the sal-lacZ and vgQE-lacZ reporters in wing discs during the third instar larval stage, as well as the wing and eye phenotypes in adult flies.
The effects of depleting different Mediator subunits on the expression of the <i>sal-lacZ</i> and <i>vgQE-lacZ</i> reporters in wing discs during the third instar larval stage, as well as the wing and eye phenotypes in adult flies.

CDK9 and Yorkie also positively regulate the Mad/Smad-dependent transcription

Besides CDK8, several other kinases, such as CDK7, CDK9, GSK3 (Glycogen synthase kinase 3), and MAPKs (mitogen-activated protein kinases) such as ERK (extracellular signal-regulated kinase) and ERK2, have been implicated to phosphorylate and regulate the transcriptional activity of Smads [14,15,68,71] (Fig 8A, see below). The four phosphorylation sites (Ser or Thr residues) within the linker region of Smads appear to be conserved from Drosophila to mammals (Fig 8B; see Discussion). The phosphorylation of Smads within the linker region may facilitate the subsequent binding with transcription co-factors, such as YAP (Yes-associated protein) [14]. However, it is still unclear whether all of these kinases regulate Smads activity in vivo. With the exception of YAP (Yorkie or Yki, in Drosophila), it is also unclear whether these regulatory mechanisms are conserved during evolution.

Validation of additional transcriptional cofactors for their roles in regulating Mad-dependent transcription.
Fig. 8. Validation of additional transcriptional cofactors for their roles in regulating Mad-dependent transcription.
(A) Model: linker region of pMad may be phosphorylated by CDK8, CDK9, or MAPKs as priming kinase recruiting Yki/YAP binding to pMad to drive target gene, such as sal transcription; and further phosphorylation by Sgg/GSK3 at the linker region may switch the binding to dSmuf1 and causes pMad degradation. (B) Sequence alignment of part of the Mad/Smad1 linker region from different species showing the conservation of the potential phosphorylation sites by CDKs, MAPKs, and GSK3. Representative confocal images of anti-β-Gal staining of wing discs of the following genotypes: (C) ap-Gal4, sal-lacZ/+; UAS-yki-RNAi/+; (D) ap-Gal4, sal-lacZ/UAS-Cdk9-RNAi; (E) ap-Gal4, sal-lacZ/+; UAS-CycT-RNAi/+; (F) ap-Gal4, sal-lacZ/UAS-Cdk7-RNAi; (G) ap-Gal4, sal-lacZ/+; UAS-rl-RNAi/+; (H) ap-Gal4, sal-lacZ/+; UAS-ERK2-RNAi/+; and (I) ap-Gal4, sal-lacZ/UAS-sgg-RNAi. Scale bar in D: 25μm. (J) Quantification of Sal-lacZ expression. The grey columns represent the average of Sal-lacZ expression in the ventral compartment of the indicated genotypes, and light green columns represent the measurements in the corresponding dorsal compartments. N = 5 for the quantification of sal-lacZ expression after depleting Yki, Cdk9, or CycT in the dorsal compartment; N = 3 for the quantification of sal-lacZ expression after depleting Cdk7 or Sgg in the dorsal compartment. At least five wing discs were examined for depletion of Rl (G) and ERK2 (H), and the represented images were shown.

To validate the relevance of these kinases in regulating Mad-dependent gene expression, we depleted the Drosophila orthologs of CDK7, CDK9, Shaggy (Sgg, the GSK3 homolog in Drosophila), Rolled, and dERK2 (MAPK/ERK homologs in Drosophila), in the dorsal compartment of wing discs (using ap-Gal4 as above), and then analyzed sal-lacZ expression in the wing pouch. As expected for a positive role of Yki in regulated Mad-dependent transcription [14], depletion of Yki in the dorsal cells significantly reduced the expression of sal-lacZ compared to the cells in the ventral compartment of the same discs (Fig 8C and 8J). Using the same approach, we have observed that depleting CDK9 (Fig 8D and 8J) and its partner CycT (Cyclin T, Fig 8E and 8J; [72]) also reduced sal-lacZ expression. These observations suggest that both Yki and CDK9-CycT are required for Mad/Smad-dependent transcription in Drosophila, which is consistent to the previous reports [14,31]. However, depletion of CDK7 (Fig 8F and Fig 8J) or Drosophila MAPK homologs, either Rolled (Fig 8G) or dERK2 (Fig 8H), did not affect the expression of sal-lacZ. Although depletion of Sgg increased the size of the dorsal compartment, the intensity of anti-β-Gal staining remained similar to the ventral compartment (Fig 8I and 8J). We note that depleting CDK9 (S7D Fig), Med12 (S7E Fig), or Med13 (S7F Fig) have no obvious effects on the expression of UAS-GFP reporter, suggesting that their effects on sal-lacZ expression are independent of the Gal4 activity per se. Together with the previous reports [14,68], our in vivo analyses have validated the conserved roles of CDK8-CycC, CDK9-CycT, and Yki/YAP on Mad/Smad-dependent transcription.

Discussion

To study the function and regulation of CDK8 in vivo, we have developed a genetic system 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 us to identify multiple components of the Dpp/TGFβ signaling pathway that can genetically interact with the CDK8-CycC complex in vivo. Our 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 our understanding of the conserved function and regulation of CDK8 during development.

Multiple subunits of the Mediator complex are required for Mad/Smad-dependent transcription

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 [4,69,73]. 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 [74]. 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 [75]. 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 [70].

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 [69]. 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 [69]. In Drosophila, loss or reduction of Med15 reduced the expression of Dpp targets, resulting in smaller wings and disrupted vein patterning (mainly L2) [70]. We 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.

Aside from Med15 and CDK8, it remains unclear whether other Mediator subunits are also involved in Mad/Smad-dependent transcription. We identified six additional Mediator subunits that are required for the Mad-dependent transcription, including CycC, Med12, Med13, Med23, Med24, and Med31 (Fig 5, Fig 6, Fig 7 and Table 3). 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 [5]. The similar effects of the four CKM subunits on Mad-activity suggest that they may function together to stimulate Mad-dependent transcription. We note that depletion of seven Mediator subunits, Med7, Med8, Med14, Med16, Med17, Med21, and Med22, severely disrupts the morphology of the wing discs (Fig 7I and S10 Fig), 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 [5]. 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 (Table 3). Med1 and Med25 are loosely associated to the small Mediator complex in human cell lines [5]. 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 (Table 3). Further analyses are necessary to validate these negative data in the future. Taken together, our results indicate that not all Mediator subunits are required for the expression of the Mad-target genes that we tested in the developing wing discs.

Role of Yki/YAP and different kinases in regulating Mad/Smad-dependent transcription

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 [76]. Med23 was also reported to regulate Yki-dependent transcription of Diap1 in wing discs [77]. In this work, we found that 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.

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 [14]. Of these sites, S206 and S214 are both conserved from Drosophila to humans (Fig 8B). 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 [71]. 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 [15]. These studies suggest the following model in explaining how Smads activate the expression of their target genes and how this process is turned off (Fig 8A, Fig 9): 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 [14], 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 [14,15,31,55,68].

Working model.
Fig. 9. Working model.
Model of Mad/Smad-dependent transcription activation through the CKM and the Mediator complex. GTFs, General Transcription Factors; MH1, Mad homology 1; MH2, Mad homology 2.

Although this model (Fig 9) 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 [78]. 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 [54,7981].

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 [59,62]. 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 [14,82]. Depleting CDK8 does not affect the phosphorylation of Mad at its C-terminus as revealed by pMad immunostaining (S8 Fig), 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.

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, we did not observe any effects of depletion of CDK7 or MAPKs on sal-lacZ expression, 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 [14,15,31,55,68].

Together with the previous reports [14,15,31,55,68,83], our data support that CDK8-CycC and CDK9-CycT may phosphorylate pMad at the linker region, which may facilitate the binding between Yki and Mad. We speculate 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 (Fig 9). 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 (Fig 9).

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 [84]. Thus, it will be important to test whether additional Mediator subunits that we 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.

Potential role of CDK8-CycC in regulating cross-talks among different signaling pathways

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 [10,11], 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 [55,59,60,62]. 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.

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 (Fig 1). Our analyses on the genetic interactions between CDK8 and the components of the Dpp signaling pathway led us to discover the role of the Mediator complex in Mad-stimulated transcription of sal. However, there is a gap in our understanding of how 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 [85]. 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 [61,86]. 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 [61]. 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.

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 [12], Mad/Smad proteins ([14,15] and this work). 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 [87]. In mammalian cells, Med12 is also known to regulate the activities of Gli proteins, the key transcription factors downstream of Hh signaling [88,89]. 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 [75]. 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, we speculate 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.

Identification of novel genomic loci that genetically interact with CDK8 in vivo

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 [5], Drosophila serves as an ideal model system to identify both the upstream regulators and the downstream effectors of CDK8 activity in vivo. Our 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 us to identify 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 our understanding of how dysregulation of CDK8 contributes to human diseases, thereby aiding the development of therapeutic approaches.

Materials and methods

Fly strains

Flies were raised on a standard cornmeal, molasses and yeast medium, and all genetic crosses were maintained at 25˚C. The UAS-Cdk8+ and UAS-Cdk8KD lines were generated using the pUASt vector [36]. The construct allowing conditional expression of a kinase-dead CDK8 form (D173A; [90]) was generated through site-specific mutagenesis by double PCR, using the overlap extension method. The UAS-Cdk8-RNAi and UAS-CycC-RNAi lines were generated using the pVALIUM20 vector [91], and the UAS-Cdk8-RNAi CycC-RNAi line was generated using the pNP vector [92]. The vgQE-lacZ line was received from Gary Struhl [66,67].

We obtained the following strains from the Bloomington Drosophila Stock Center: ap-Gal4 (BL-3041), nub-Gal4 (BL-25754), sal-lacZ (BL-11340), UAS-Cdk7-RNAi (BL-57245), UAS-Cdk9-RNAi (BL-34982), UAS-CycT-RNAi (BL-32976), UAS-dpp-RNAi (BL-33618), UAS-2xEGFP (BL-6874), UAS-erk-RNAi (BL-34744), UAS-Mad-RNAi (BL-31315), UAS-Mad-RNAi (BL-43183), UAS-Medea-RNAi (BL-43961), UAS-rl-RNAi (BL-34855), UAS-sgg-RNAi (BL-38293), UAS-yki-RNAi (BL-34067), and all deficiency (Df) lines (S1 Table). Of the two transgenic RNAi lines targeting Mad, the BL-31315 line (S4B and S4B’ Fig, Fig 6B) generated stronger effects than the BL-43183 line (e.g., Fig 5B) when expressed using the ap-Gal4 driver. In addition, we tested the following mutant alleles of the Dpp signaling pathway: Dadj1E4/TM3, Sb1 (BL-10305), DadMI04922/TM3 Sb1, Ser1 (BL-37913), dppd6/CyO (BL-2062), dpphr92/SM6a (BL-2069), dpps11/CyO (BL-2065), Mad1-2/CyO (BL-7323), Mad12/CyO (BL-58785), Mad8-2/CyO (BL-7324), Madk00237/CyO (BL-10474), MadKG00581/CyO (BL-14578), Medea1/TM3 Sb1, Ser1 (BL-9033), Medea13/TM3 Sb1 (BL-7340), tkv7/CyO (BL-3242), and tkvk16713/CyO (BL-11191).

The following RNAi stocks, generated by the Drosophila TRiP project [91], were used to deplete the subunits of the Mediator complex: UAS-Med1-RNAi (BL-34662), UAS-Med4-RNAi (BL-34697), UAS-Med6-RNAi/TM3 Sb1 (BL-33743), UAS-Med7-RNAi (BL-34663), UAS-Med8-RNAi (BL-34926), UAS-Med9-RNAi (BL-33678), UAS-Med10-RNAi (BL-34031), UAS-Med11-RNAi/TM3 Sb1 (BL-34083), UAS-Med12-RNAi (BL-34588), UAS-Med13-RNAi (BL-34630), UAS-Med14-RNAi (BL-34575), UAS-Med15-RNAi (BL-32517), UAS-Med16-RNAi (BL-34012), UAS-Med17-RNAi (BL-34664), UAS-Med18-RNAi (BL-42634), UAS-Med19-RNAi (BL-33710), UAS-Med20-RNAi (BL-34577), UAS-Med21-RNAi (BL-34731), UAS-Med22-RNAi (BL-34573), UAS-Med23-RNAi (BL-34658), UAS-Med24-RNAi (BL-33755), UAS-Med25-RNAi (BL-42501), UAS-Med26-RNAi (BL-28572), UAS-Med27-RNAi (BL-34576), UAS-Med28-RNAi/TM3 Sb1 (BL-32459), UAS-Med29-RNAi (BL-57259), UAS-Med30-RNAi/TM3 Sb1 (BL-36711), and UAS-Med31-RNAi (BL-34574).

To facilitate the dominant modifier genetic screen and the subsequent analyses, we generated the following strains using the standard Drosophila genetics: “w1118; nub-Gal4>UAS-Cdk8+/CyO” (i.e., “nub>Cdk8+/CyO” line), “w1118; nub-Gal4; UAS-Cdk8-RNAi” (i.e., “nub>Cdk8-i” line), “w1118; nub-Gal4; UAS-CycC-RNAi” (i.e., “nub>CycC-i” line), “w1118; nub-Gal4; UAS-Cdk8-RNAi CycC-RNAi” (i.e., “nub>Cdk8-i CycC-i” line), and “w1118; ap-Gal4, sal-lacZ/T(2:3)”.

For the Df lines in the X chromosome, we crossed Df female virgins with males of with the “nub>Cdk8+/CyO”, “nub>Cdk8-i”, “nub>CycC-i”, or “nub>Cdk8-i CycC-i” stocks. For the Df lines in the second and third chromosomes, the Df males were crossed with female virgins of the afore-described stocks carrying the CDK8-specific phenotypes. The control crosses were performed using w1118 males and female virgins. For each of these crosses, the wing vein patterns in ~10 F1 females without any balancer chromosomes were inspected under dissecting microscopes for potential dominant modifications. With few exceptions (S1 Table), the wing vein phenotypes and dominant modifications are generally stereotypical with high penetrance. For instance, we crossed Df(1)BSC531, w1118/FM7h female virgins with “w1118/Y; nub>Cdk8+/CyO” males, and then scored F1 females with the following genotype: “w1118, Df(1)BSC531/ w1118; nub>Cdk8+/+”. Similarly, we crossed “w1118; nub-Gal4; UAS-Cdk8-RNAi” female virgins with “Df(2R)Exel6064/CyO” males, and then scored F1 females with the following genotype: “w1118/+; nub-Gal4/Df(2R)Exel6064; UAS-Cdk8-RNAi/+”. Df lines that caused lethality in F1 were considered as the enhancers.

Adult Drosophila wing imaging

The wings from adult females were dissected onto slides, briefly washed using isopropanol, and then mounted in 50% Canada balsam diluted in isopropanol. Images were taken under 5X objective of a microscope (Leica DM2500) and then processed by Adobe Photoshop CS6 software.

Immunocytochemistry

Wing discs from third instar larvae at the late wandering stage were dissected and fixed in 5% formaldehyde at room temperature for 30 minutes. After rinsing with PBS-Triton X-100 (0.2%), the samples were blocked in PBS-Triton X-100-NGS-BSA (PBS+0.2% Triton X-100+5% Normal Goat Serum+0.2% Bovine Serum Albumin) at room temperature for one hour. For immunostaining of Drosophila CDK8 and CycC, we used anti-dCDK8 (1:2000) and anti-dCycC (1:2000) antibodies [9395], diluted in PBS-Triton X-100-NGS-BSA. Expression of the lacZ reporter expression was detected using an anti-β-galactosidase monoclonal antibody (1:50 in PBS-Triton X-100-NGS-BSA; obtained from the Developmental Studies Hybridoma Bank, DSHB-40-1a-s). C-terminal phosphorylated Mad (equivalent sites to human Smad3 S423+S425) was detected by anti-pSmad3 (1:500 in PBS-Triton X-100-NGS-BSA; purchased from Abcam, ab118825). Wing discs were incubated with these primary antibodies overnight at 4˚C on a rotator. After rinsing with PBS-Triton X-100, the discs were then incubated with the fluorophore conjugated secondary antibodies: goat anti-guinea pig (106-545-003), goat anti-mouse (115-545-003), or goat anti-rabbit (111-545-003) (all purchased from Jackson Immunological Laboratories). These secondary antibodies were diluted 1:1000 in PBS-Triton X-100-NGS-BSA, and incubated with the samples for one hour at room temperature. Discs were then stained with 1 μM DAPI at room temperature for 10 minutes, rinsed two more times with PBS-Triton X-100, and mounted in the Vectashield mounting media (Vector Laboratories, H-1000). Confocal images were taken with a Nikon Ti Eclipse confocal microscope system, with images processed using the Adobe Photoshop CS6 software.

Quantification of anti-β-galactosidase was performed with Nikon NIS software and Microsoft Excel: a single section of the wing discs was selected for the following quantification based on the DAPI channel, which indicates the cell nucleus are on the same focal plat. Three lines around 50μm long, 10–15μm apart, were drawn along the dorsal-ventral boundary. The line-scan profile of intensity for each line was calculated along each line (S5A and S5B Fig; genotype: ap-Gal4, sal-lacZ/+; UAS-Cdk8-i/+). The area below the intensity index profile represents the Sal-lacZ expression levels along the line (S5B Fig). To obtain the average intensity of dorsal or ventral compartment, the dorsal or ventral compartment index area was divided by the dorsal or ventral length of the line (S5C Fig). The intensity for three lines was normalized and averaged in dorsal and ventral compartments (S5C Fig, S5D Fig, S2 Table). Following this approach, five wing discs for each genotype were analyzed to quantify the expression of Sal-lacZ in dorsal and ventral compartments, and statistical significance was calculated using Student’s one-tailed t-test (S5E Fig, S2 Table).

To validate the afore described quantification method, we also measured the signaling intensity by selecting 20x20μm squares in the dorsal and the ventral compartments of the same wing disc using the Nikon NIS software (S6A and S6B Fig; genotype: ap-Gal4, sal-lacZ/+; UAS-Med15-i/+). We then calculated the dorsal to ventral ratios of the signal intensities of three different discs (S6B Fig), followed by statistical analyses using the Student’s one-tailed t-test (S6C and S6D Fig). We obtained similar results to the quantification based on the line profiles as described above.

GST-pull down assay

Full-length CDK8 fused with a N-terminal GST tag was described previously [36]. The primers Mad-5.1 (F: 5’-caccATGGACACCGACGATGTGGA-3’) and Mad-3.3 (F: 5’-ctaTTAGGATACCGAACTAATTG-3’) were used for full-length Mad (AA1-455), Mad-5.1 and Mad-3.1 (F: 5’-ctaCGGGAGCACCGGACTCTCCA-3’) were used for a “Mad-N1” fragment (AA1-150) that contains MH1 domain (AA10-133), Mad-5.1 and Mad-3.2 (F: 5’-ctaATCCTCCGAGGGACTGTAGG-3’) were used for the “Mad-N2” fragment (AA1-230) that contains the MH1 domain and part of the linker region, Mad-5.2 (F: 5’-caccatgCCAGTACTCGTTCCTCGCCA-3’) and Mad-3.3 were used for the “Mad-C2” fragment (AA151-455) that contains the MH2 domain (AA255-455) and part of the linker region, and Mad-5.3 (F: 5’-caccatgGGCAACTCCAACAATCCGAA-3’) and Mad-3.3 were for the “Mad-C1” fragment (AA231-455) that contains the MH2 domain. These coding sequences were amplified from a cDNA clone of the Mad gene (LD12679) using PrimeStar Max premix (Takara, R045A). The amplified products were inserted into the pENTR/D-TOPO vector (ThermoFisher, K240020) and recombined into the pDEST17 vector (N-terminal 6XHis tag) using the Gateway LR Clonase II Enzyme mix (ThermoFisher, 11791100) in E. coli strain DH5α. The constructs were transformed to E. coli strain Rosetta, received from Craig Kaplan, for protein expression using standard protocols.

GST or GST-CDK8 was purified with Glutathione Sepharose 4B (GE Healthcare, 17-0756-01) beads with standard purification protocol. After a final wash, the buffer was replaced by the GST pull-down buffer (20mM Tris-HCl pH 7.5, 10mM MgCl2, 100mM NaCl, 1mM DTT, 0.1% NP-40). His-tagged Mad fragments were extracted from the pull-down buffer by sonication. 50μL GST or GST-CDK8 coated beads (0.5–1μg protein) was mixed with 500μL of Mad fragments cell lysate and incubated at 4°C for 3 hours. These samples were then washed with 1mL pull-down buffer at 4°C for 5 times, 1 minute each. The interaction was detected by Western Blot with the primary antibody, anti-His (1:3000; Sigma, H1029), and the secondary antibody, anti-mouse (1:2000; Jackson Immunological Laboratories, 115-035-174).

Yeast two-hybrid (Y2H) assay

Full-length CDK8 was amplified from a pBS-CDK8 cDNA clone using primers CDK8-5.1 (F: 5’-caccATGGACTACG ATTTCAAGAT-3’) and CDK8-3.1 (F: 5’-TCAGTTGAAGCGCTGGAAGT-3’), and then inserted into the pENTR/D-TOPO vector. The Gateway LR Clonase II Enzyme mix was used to recombine CDK8 cDNA into the pGADT7-GW (prey) vector, a gift from Yuhai Cui (Addgene plasmid # 61702) [96]. The linker region of Mad was amplified with Mad-5.2 and Mad-3.2 primers from a cDNA clone of the Mad gene (LD12679) using PrimeStar Max premix and inserted into the pENTR/D-TOPO vector. All pENTR Mad fragments were recombined into the pGBKT7-GW (bait) vector, a gift from Yuhai Cui (Addgene plasmid # 61703) [96], using the Gateway LR Clonase II Enzyme mix. The Y2H assay was performed using the AH109 yeast strain, as described previously [96].

Statistical analysis

Standard deviation and Student’s one-tailed t-tests were performed using Microsoft Excel. Statistical significance (* p<0.05; ** p<0.01; *** p<0.001) was shown in figures and all error bars indicate standard deviation.

Supporting information

S1 Fig [a]
Effects of CDK8 on the size of wings, cell number, and cell sizes.

S2 Fig [a]
Effects of CDK8 on the wing morphology with .

S3 Fig [a]
Effects of Dpp signaling pathway components on .

S4 Fig [a]
Validation of the reporter.

S5 Fig [a]
Quantification of the expression.

S6 Fig [a]
An alternative method to quantify the expression.

S7 Fig [green]
Effects of depleting subunits of the CKM and CDK9 on , and effect of depleting Ap on expression.

S8 Fig [a]
Depletion of CDK8 or CycC does not affect the levels of p-Mad.

S9 Fig [fl]
Additional results from the Y2H assay.

S10 Fig [a]
Depletion of dedicated Mediator subunits strongly disrupted wing disc morphology.

S1 Table [xlsx]
Results of 490 deficiency () lines tested for potential dominant modification of vein phenotypes caused by altered levels of CDK8 or CycC.

S2 Table [xlsx]
Quantification of the Sal-lacZ expression.


Zdroje

1. Boube M, Joulia L, Cribbs DL, Bourbon HM (2002) Evidence for a mediator of RNA polymerase II transcriptional regulation conserved from yeast to man. Cell 110: 143–151. doi: 10.1016/s0092-8674(02)00830-9 12150923

2. Bourbon HM, Aguilera A, Ansari AZ, Asturias FJ, Berk AJ, et al. (2004) A unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II. Mol Cell 14: 553–557. doi: 10.1016/j.molcel.2004.05.011 15175151

3. Kornberg RD (2005) Mediator and the mechanism of transcriptional activation. Trends Biochem Sci 30: 235–239. doi: 10.1016/j.tibs.2005.03.011 15896740

4. Soutourina J (2018) Transcription regulation by the Mediator complex. Nat Rev Mol Cell Biol 19: 262–274. doi: 10.1038/nrm.2017.115 29209056

5. Bourbon HM (2008) Comparative genomics supports a deep evolutionary origin for the large, four-module transcriptional mediator complex. Nucleic Acids Res 36: 3993–4008. doi: 10.1093/nar/gkn349 18515835

6. Conaway RC, Conaway JW (2011) Function and regulation of the Mediator complex. Curr Opin Genet Dev 21: 225–230. doi: 10.1016/j.gde.2011.01.013 21330129

7. Fondell JD (2013) The Mediator complex in thyroid hormone receptor action. Biochim Biophys Acta 1830: 3867–3875. doi: 10.1016/j.bbagen.2012.02.012 22402254

8. Poss ZC, Ebmeier CC, Taatjes DJ (2013) The Mediator complex and transcription regulation. Crit Rev Biochem Mol Biol 48: 575–608. doi: 10.3109/10409238.2013.840259 24088064

9. Yin JW, Wang G (2014) The Mediator complex: a master coordinator of transcription and cell lineage development. Development 141: 977–987. doi: 10.1242/dev.098392 24550107

10. Morris EJ, Ji JY, Yang F, Di Stefano L, Herr A, et al. (2008) E2F1 represses beta-catenin transcription and is antagonized by both pRB and CDK8. Nature 455: 552–556. doi: 10.1038/nature07310 18794899

11. Zhao J, Ramos R, Demma M (2013) CDK8 regulates E2F1 transcriptional activity through S375 phosphorylation. Oncogene 32: 3520–3530. doi: 10.1038/onc.2012.364 22945643

12. Fryer CJ, White JB, Jones KA (2004) Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover. Mol Cell 16: 509–520. doi: 10.1016/j.molcel.2004.10.014 15546612

13. Donner AJ, Szostek S, Hoover JM, Espinosa JM (2007) CDK8 is a stimulus-specific positive coregulator of p53 target genes. Mol Cell 27: 121–133. doi: 10.1016/j.molcel.2007.05.026 17612495

14. Alarcon C, Zaromytidou AI, Xi Q, Gao S, Yu J, et al. (2009) Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-beta pathways. Cell 139: 757–769. doi: 10.1016/j.cell.2009.09.035 19914168

15. Aleman A, Rios M, Juarez M, Lee D, Chen A, et al. (2014) Mad linker phosphorylations control the intensity and range of the BMP-activity gradient in developing Drosophila tissues. Sci Rep 4: 6927. doi: 10.1038/srep06927 25377173

16. Zhao X, Feng D, Wang Q, Abdulla A, Xie XJ, et al. (2012) Regulation of lipogenesis by cyclin-dependent kinase 8-mediated control of SREBP-1. J Clin Invest 122: 2417–2427. doi: 10.1172/JCI61462 22684109

17. Bancerek J, Poss ZC, Steinparzer I, Sedlyarov V, Pfaffenwimmer T, et al. (2013) CDK8 Kinase Phosphorylates Transcription Factor STAT1 to Selectively Regulate the Interferon Response. Immunity 38: 250–262. doi: 10.1016/j.immuni.2012.10.017 23352233

18. Clark AD, Oldenbroek M, Boyer TG (2015) Mediator kinase module and human tumorigenesis. Crit Rev Biochem Mol Biol 50: 393–426. doi: 10.3109/10409238.2015.1064854 26182352

19. Xu W, Ji JY (2011) Dysregulation of CDK8 and Cyclin C in tumorigenesis. J Genet Genomics 38: 439–452. doi: 10.1016/j.jgg.2011.09.002 22035865

20. Schiano C, Casamassimi A, Rienzo M, de Nigris F, Sommese L, et al. (2014) Involvement of Mediator complex in malignancy. Biochim Biophys Acta 1845: 66–83. doi: 10.1016/j.bbcan.2013.12.001 24342527

21. Spaeth JM, Kim NH, Boyer TG (2011) Mediator and human disease. Semin Cell Dev Biol 22: 776–787. doi: 10.1016/j.semcdb.2011.07.024 21840410

22. Li X, Liu M, Ji JY (2019) Understanding Obesity as a Risk Factor for Uterine Tumors Using Drosophila. Adv Exp Med Biol 1167: 129–155. doi: 10.1007/978-3-030-23629-8_8 31520353

23. Firestein R, Bass AJ, Kim SY, Dunn IF, Silver SJ, et al. (2008) CDK8 is a colorectal cancer oncogene that regulates beta-catenin activity. Nature 455: 547–551. doi: 10.1038/nature07179 18794900

24. Kapoor A, Goldberg MS, Cumberland LK, Ratnakumar K, Segura MF, et al. (2010) The histone variant macroH2A suppresses melanoma progression through regulation of CDK8. Nature 468: 1105–1109. doi: 10.1038/nature09590 21179167

25. Broude EV, Gyorffy B, Chumanevich AA, Chen M, McDermott MS, et al. (2015) Expression of CDK8 and CDK8-interacting Genes as Potential Biomarkers in Breast Cancer. Curr Cancer Drug Targets 15: 739–749. doi: 10.2174/156800961508151001105814 26452386

26. Brewster CD, Birkenheuer CH, Vogt MB, Quackenbush SL, Rovnak J (2011) The retroviral cyclin of walleye dermal sarcoma virus binds cyclin-dependent kinases 3 and 8. Virology 409: 299–307. doi: 10.1016/j.virol.2010.10.022 21067790

27. Rovnak J, Quackenbush SL (2002) Walleye dermal sarcoma virus cyclin interacts with components of the mediator complex and the RNA polymerase II holoenzyme. J Virol 76: 8031–8039. doi: 10.1128/jvi.76.16.8031-8039.2002 12134008

28. Xu W, Wang Z, Zhang W, Qian K, Li H, et al. (2015) Mutated K-ras activates CDK8 to stimulate the epithelial-to-mesenchymal transition in pancreatic cancer in part via the Wnt/beta-catenin signaling pathway. Cancer Lett 356: 613–627. doi: 10.1016/j.canlet.2014.10.008 25305448

29. Osherovich L (2008) CDK8 is enough in colorectal cancer Science-Business eXchange 1: 5–7.

30. Rzymski T, Mikula M, Wiklik K, Brzozka K (2015) CDK8 kinase—An emerging target in targeted cancer therapy. Biochim Biophys Acta 1854: 1617–1629. doi: 10.1016/j.bbapap.2015.05.011 26006748

31. Aragon E, Goerner N, Zaromytidou AI, Xi Q, Escobedo A, et al. (2011) A Smad action turnover switch operated by WW domain readers of a phosphoserine code. Genes Dev 25: 1275–1288. doi: 10.1101/gad.2060811 21685363

32. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401–415. 8223268

33. Duffy JB (2002) GAL4 system in Drosophila: a fly geneticist's Swiss army knife. Genesis 34: 1–15. doi: 10.1002/gene.10150 12324939

34. Kambadur R, Koizumi K, Stivers C, Nagle J, Poole SJ, et al. (1998) Regulation of POU genes by castor and hunchback establishes layered compartments in the Drosophila CNS. Genes Dev 12: 246–260. doi: 10.1101/gad.12.2.246 9436984

35. Milan M, Campuzano S, Garcia-Bellido A (1997) Developmental parameters of cell death in the wing disc of Drosophila. Proc Natl Acad Sci U S A 94: 5691–5696. doi: 10.1073/pnas.94.11.5691 9159134

36. Loncle N, Boube M, Joulia L, Boschiero C, Werner M, et al. (2007) Distinct roles for Mediator Cdk8 module subunits in Drosophila development. EMBO J 26: 1045–1054. doi: 10.1038/sj.emboj.7601566 17290221

37. Bridges CB (1919) Specific modifiers of eosin eye color in Drosophila melanogaster. J Exp Zool 28: 337–384.

38. St Johnston D (2002) The art and design of genetic screens: Drosophila melanogaster. Nat Rev Genet 3: 176–188. doi: 10.1038/nrg751 11972155

39. Ji JY, Haghnia M, Trusty C, Goldstein LS, Schubiger G (2002) A genetic screen for suppressors and enhancers of the Drosophila cdk1-cyclin B identifies maternal factors that regulate microtubule and microfilament stability. Genetics 162: 1179–1195. 12454065

40. Lee LA, Elfring LK, Bosco G, Orr-Weaver TL (2001) A genetic screen for suppressors and enhancers of the Drosophila PAN GU cell cycle kinase identifies cyclin B as a target. Genetics 158: 1545–1556. 11514446

41. Kennison JA, Tamkun JW (1988) Dosage-dependent modifiers of polycomb and antennapedia mutations in Drosophila. Proc Natl Acad Sci U S A 85: 8136–8140. doi: 10.1073/pnas.85.21.8136 3141923

42. Ji JY, Miles WO, Korenjak M, Zheng Y, Dyson NJ (2012) In vivo regulation of E2F1 by Polycomb group genes in Drosophila. G3 (Bethesda) 2: 1651–1660.

43. Nadeau JH (2003) Modifier genes and protective alleles in humans and mice. Curr Opin Genet Dev 13: 290–295. doi: 10.1016/s0959-437x(03)00061-3 12787792

44. Nadeau JH (2001) Modifier genes in mice and humans. Nat Rev Genet 2: 165–174. doi: 10.1038/35056009 11256068

45. Parks AL, Cook KR, Belvin M, Dompe NA, Fawcett R, et al. (2004) Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome. Nat Genet 36: 288–292. doi: 10.1038/ng1312 14981519

46. Cook RK, Christensen SJ, Deal JA, Coburn RA, Deal ME, et al. (2012) The generation of chromosomal deletions to provide extensive coverage and subdivision of the Drosophila melanogaster genome. Genome Biol 13: R21. doi: 10.1186/gb-2012-13-3-r21 22445104

47. Nagarkar-Jaiswal S, Lee PT, Campbell ME, Chen K, Anguiano-Zarate S, et al. (2015) A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila. Elife 4.

48. Tsuneizumi K, Nakayama T, Kamoshida Y, Kornberg TB, Christian JL, et al. (1997) Daughters against dpp modulates dpp organizing activity in Drosophila wing development. Nature 389: 627–631. doi: 10.1038/39362 9335506

49. Hamaratoglu F, Affolter M, Pyrowolakis G (2014) Dpp/BMP signaling in flies: from molecules to biology. Semin Cell Dev Biol 32: 128–136. doi: 10.1016/j.semcdb.2014.04.036 24813173

50. Affolter M, Basler K (2007) The Decapentaplegic morphogen gradient: from pattern formation to growth regulation. Nat Rev Genet 8: 663–674. doi: 10.1038/nrg2166 17703237

51. Upadhyay A, Moss-Taylor L, Kim MJ, Ghosh AC, O'Connor MB (2017) TGF-beta Family Signaling in Drosophila. Cold Spring Harb Perspect Biol 9.

52. Massague J (2012) TGFbeta signalling in context. Nat Rev Mol Cell Biol 13: 616–630. doi: 10.1038/nrm3434 22992590

53. Raftery LA, Sutherland DJ (1999) TGF-beta family signal transduction in Drosophila development: from Mad to Smads. Dev Biol 210: 251–268. doi: 10.1006/dbio.1999.9282 10357889

54. Santibanez JF, Krstic J., Quintanilla M., Bernabeu C. (2016) TGF–β Signalling and Its Role in Cancer Progression and Metastasis. eLS.

55. Restrepo S, Zartman JJ, Basler K (2014) Coordination of patterning and growth by the morphogen DPP. Curr Biol 24: R245–255. doi: 10.1016/j.cub.2014.01.055 24650915

56. Affolter M, Marty T, Vigano MA, Jazwinska A (2001) Nuclear interpretation of Dpp signaling in Drosophila. EMBO J 20: 3298–3305. doi: 10.1093/emboj/20.13.3298 11432817

57. Moustakas A, Souchelnytskyi S, Heldin CH (2001) Smad regulation in TGF-beta signal transduction. J Cell Sci 114: 4359–4369. 11792802

58. Malik S, Roeder RG (2005) Dynamic regulation of pol II transcription by the mammalian Mediator complex. Trends Biochem Sci 30: 256–263. doi: 10.1016/j.tibs.2005.03.009 15896744

59. Blair SS (2007) Wing vein patterning in Drosophila and the analysis of intercellular signaling. Annu Rev Cell Dev Biol 23: 293–319. doi: 10.1146/annurev.cellbio.23.090506.123606 17506700

60. De Celis JF (2003) Pattern formation in the Drosophila wing: The development of the veins. Bioessays 25: 443–451. doi: 10.1002/bies.10258 12717815

61. de Celis JF, Barrio R (2000) Function of the spalt/spalt-related gene complex in positioning the veins in the Drosophila wing. Mech Dev 91: 31–41. doi: 10.1016/s0925-4773(99)00261-0 10704828

62. Crozatier M, Glise B, Vincent A (2004) Patterns in evolution: veins of the Drosophila wing. Trends Genet 20: 498–505. doi: 10.1016/j.tig.2004.07.013 15363904

63. Spradling AC, Stern D, Beaton A, Rhem EJ, Laverty T, et al. (1999) The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes. Genetics 153: 135–177. 10471706

64. Treisman JE, Rubin GM (1996) Targets of glass regulation in the Drosophila eye disc. Mech Dev 56: 17–24. doi: 10.1016/0925-4773(96)00508-4 8798144

65. Nellen D, Burke R, Struhl G, Basler K (1996) Direct and long-range action of a DPP morphogen gradient. Cell 85: 357–368. doi: 10.1016/s0092-8674(00)81114-9 8616891

66. Zecca M, Struhl G (2007) Control of Drosophila wing growth by the vestigial quadrant enhancer. Development 134: 3011–3020. doi: 10.1242/dev.006445 17634191

67. Zecca M, Struhl G (2007) Recruitment of cells into the Drosophila wing primordium by a feed-forward circuit of vestigial autoregulation. Development 134: 3001–3010. doi: 10.1242/dev.006411 17634192

68. Xu P, Lin X, Feng XH (2016) Posttranslational Regulation of Smads. Cold Spring Harb Perspect Biol 8.

69. Kato Y, Habas R, Katsuyama Y, Naar AM, He X (2002) A component of the ARC/Mediator complex required for TGF beta/Nodal signalling. Nature 418: 641–646. doi: 10.1038/nature00969 12167862

70. Terriente-Felix A, Lopez-Varea A, de Celis JF (2010) Identification of genes affecting wing patterning through a loss-of-function mutagenesis screen and characterization of med15 function during wing development. Genetics 185: 671–684. doi: 10.1534/genetics.109.113670 20233856

71. Fuentealba LC, Eivers E, Ikeda A, Hurtado C, Kuroda H, et al. (2007) Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal. Cell 131: 980–993. doi: 10.1016/j.cell.2007.09.027 18045539

72. Bacon CW, D'Orso I (2019) CDK9: a signaling hub for transcriptional control. Transcription 10: 57–75. doi: 10.1080/21541264.2018.1523668 30227759

73. Naar AM, Lemon BD, Tjian R (2001) Transcriptional coactivator complexes. Annu Rev Biochem 70: 475–501. doi: 10.1146/annurev.biochem.70.1.475 11395415

74. Allen BL, Taatjes DJ (2015) The Mediator complex: a central integrator of transcription. Nat Rev Mol Cell Biol 16: 155–166. doi: 10.1038/nrm3951 25693131

75. Stevens JL, Cantin GT, Wang G, Shevchenko A, Berk AJ (2002) Transcription control by E1A and MAP kinase pathway via Sur2 mediator subunit. Science 296: 755–758. doi: 10.1126/science.1068943 11934987

76. Galli GG, Carrara M, Yuan WC, Valdes-Quezada C, Gurung B, et al. (2015) YAP Drives Growth by Controlling Transcriptional Pause Release from Dynamic Enhancers. Mol Cell 60: 328–337. doi: 10.1016/j.molcel.2015.09.001 26439301

77. Oh H, Slattery M, Ma L, Crofts A, White KP, et al. (2013) Genome-wide association of Yorkie with chromatin and chromatin-remodeling complexes. Cell Rep 3: 309–318. doi: 10.1016/j.celrep.2013.01.008 23395637

78. Tansey WP (2001) Transcriptional activation: risky business. Genes Dev 15: 1045–1050. doi: 10.1101/gad.896501 11331599

79. Cantelli G, Crosas-Molist E, Georgouli M, Sanz-Moreno V (2017) TGFBeta-induced transcription in cancer. Semin Cancer Biol 42: 60–69. doi: 10.1016/j.semcancer.2016.08.009 27586372

80. Kahata K, Dadras MS, Moustakas A (2018) TGF-beta Family Signaling in Epithelial Differentiation and Epithelial-Mesenchymal Transition. Cold Spring Harb Perspect Biol 10.

81. Yu Y, Feng XH (2019) TGF-beta signaling in cell fate control and cancer. Curr Opin Cell Biol 61: 56–63. doi: 10.1016/j.ceb.2019.07.007 31382143

82. Galbraith MD, Donner AJ, Espinosa JM (2010) CDK8: A positive regulator of transcription. Transcr 1: 4–12.

83. Eivers E, Fuentealba LC, Sander V, Clemens JC, Hartnett L, et al. (2009) Mad is required for wingless signaling in wing development and segment patterning in Drosophila. PLoS One 4: e6543. doi: 10.1371/journal.pone.0006543 19657393

84. Zhao M, Yang X, Fu Y, Wang H, Ning Y, et al. (2013) Mediator MED15 modulates transforming growth factor beta (TGFbeta)/Smad signaling and breast cancer cell metastasis. J Mol Cell Biol 5: 57–60. doi: 10.1093/jmcb/mjs054 23014762

85. de Celis JF, Barrio R (2009) Regulation and function of Spalt proteins during animal development. Int J Dev Biol 53: 1385–1398. doi: 10.1387/ijdb.072408jd 19247946

86. Organista MF, De Celis JF (2013) The Spalt transcription factors regulate cell proliferation, survival and epithelial integrity downstream of the Decapentaplegic signalling pathway. Biol Open 2: 37–48. doi: 10.1242/bio.20123038 23336075

87. Carrera I, Janody F, Leeds N, Duveau F, Treisman JE (2008) Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13. Proc Natl Acad Sci U S A 105: 6644–6649. doi: 10.1073/pnas.0709749105 18451032

88. Zhou H, Kim S, Ishii S, Boyer TG (2006) Mediator modulates Gli3-dependent Sonic hedgehog signaling. Mol Cell Biol 26: 8667–8682. doi: 10.1128/MCB.00443-06 17000779

89. Zhou H, Spaeth JM, Kim NH, Xu X, Friez MJ, et al. (2012) MED12 mutations link intellectual disability syndromes with dysregulated GLI3-dependent Sonic Hedgehog signaling. Proc Natl Acad Sci U S A 109: 19763–19768. doi: 10.1073/pnas.1121120109 23091001

90. Akoulitchev S, Chuikov S, Reinberg D (2000) TFIIH is negatively regulated by cdk8-containing mediator complexes. Nature 407: 102–106. doi: 10.1038/35024111 10993082

91. Ni JQ, Zhou R, Czech B, Liu LP, Holderbaum L, et al. (2011) A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nat Methods 8: 405–407. doi: 10.1038/nmeth.1592 21460824

92. Qiao HH, Wang F, Xu RG, Sun J, Zhu R, et al. (2018) An efficient and multiple target transgenic RNAi technique with low toxicity in Drosophila. Nat Commun 9: 4160. doi: 10.1038/s41467-018-06537-y 30297884

93. Gobert V, Osman D, Bras S, Auge B, Boube M, 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: 2837–2848. doi: 10.1128/MCB.01625-09 20368357

94. Lahue EE, Smith AV, Orr-Weaver TL (1991) A novel cyclin gene from Drosophila complements CLN function in yeast. Genes Dev 5: 2166–2175. doi: 10.1101/gad.5.12a.2166 1836192

95. Xie XJ, Hsu FN, Gao X, Xu W, Ni JQ, et al. (2015) CDK8-Cyclin C Mediates Nutritional Regulation of Developmental Transitions through the Ecdysone Receptor in Drosophila. PLoS Biol 13: e1002207. doi: 10.1371/journal.pbio.1002207 26222308

96. Lu Q, Tang X, Tian G, Wang F, Liu K, et al. (2010) Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin. Plant J 61: 259–270. doi: 10.1111/j.1365-313X.2009.04048.x 19843313


Článek vyšel v časopise

PLOS Genetics


2020 Číslo 5
Nejčtenější tento týden
Nejčtenější v tomto čísle
Kurzy Podcasty Doporučená témata Časopisy
Přihlášení
Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.

Přihlášení

Nemáte účet?  Registrujte se

#ADS_BOTTOM_SCRIPTS#