Transcription regulation by the Mediator complex.

Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.

MED12 and BRD4 cooperate to sustain cancer growth upon loss of mediator kinase.

Mediator kinases (CDK8/19) are transcriptional regulators broadly implicated in cancer. Despite their central role in fine-tuning gene-expression programs, we find complete loss of CDK8/19 is tolerated in colorectal cancer (CRC) cells. Using orthogonal functional genomic and pharmacological screens, we identify BET protein inhibition as a distinct vulnerability in CDK8/19-depleted cells. Combined CDK8/19 and BET inhibition led to synergistic growth retardation in human and mouse models of CRC. Strikingly, depletion of CDK8/19 in these cells led to global repression of RNA polymerase II (Pol II) promoter occupancy and transcription. Concurrently, loss of Mediator kinase led to a profound increase in MED12 and BRD4 co-occupancy at enhancer elements and increased dependence on BET proteins for the transcriptional output of cell-essential genes. In total, this work demonstrates a synthetic lethal interaction between Mediator kinase and BET proteins and exposes a therapeutic vulnerability that can be targeted using combination therapies.

The Mediator kinase module enhances polymerase activity to regulate transcriptional memory after heat stress in Arabidopsis.

Plants are often exposed to recurring adverse environmental conditions in the wild. Acclimation to high temperatures entails transcriptional responses, which prime plants to better withstand subsequent stress events. Heat stress (HS)-induced transcriptional memory results in more efficient re-induction of transcription upon recurrence of heat stress. Here, we identified CDK8 and MED12, two subunits of the kinase module of the transcription co-regulator complex, Mediator, as promoters of heat stress memory and associated histone modifications in Arabidopsis. CDK8 is recruited to heat-stress memory genes by HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2). Like HSFA2, CDK8 is largely dispensable for the initial gene induction upon HS, and its function in transcriptional memory is thus independent of primary gene activation. In addition to the promoter and transcriptional start region of target genes, CDK8 also binds their 3'-region, where it may promote elongation, termination, or rapid re-initiation of RNA polymerase II (Pol II) complexes during transcriptional memory bursts. Our work presents a complex role for the Mediator kinase module during transcriptional memory in multicellular eukaryotes, through interactions with transcription factors, chromatin modifications, and promotion of Pol II efficiency.

HIF1A employs CDK8-mediator to stimulate RNAPII elongation in response to hypoxia.

The transcription factor HIF1A is a key mediator of the cellular response to hypoxia. Despite the importance of HIF1A in homeostasis and various pathologies, little is known about how it regulates RNA polymerase II (RNAPII). We report here that HIF1A employs a specific variant of the Mediator complex to stimulate RNAPII elongation. The Mediator-associated kinase CDK8, but not the paralog CDK19, is required for induction of many HIF1A target genes. HIF1A induces binding of CDK8-Mediator and the super elongation complex (SEC), containing AFF4 and CDK9, to alleviate RNAPII pausing. CDK8 is dispensable for HIF1A chromatin binding and histone acetylation, but it is essential for binding of SEC and RNAPII elongation. Global analysis of active RNAPII reveals that hypoxia-inducible genes are paused and active prior to their induction. Our results provide a mechanistic link between HIF1A and CDK8, two potent oncogenes, in the cellular response to hypoxia.

Transcriptional Responses to IFN-γ Require Mediator Kinase-Dependent Pause Release and Mechanistically Distinct CDK8 and CDK19 Functions.

Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to "reprogram" cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.

The Mediator kinase module: an interface between cell signaling and transcription.

The Mediator complex controls RNA polymerase II (pol II) activity by coordinating the assembly of pol II regulatory factors at transcription start sites and by mediating interactions between enhancer-bound transcription factors (TFs) and the pol II enzyme. Mediator structure and function is completely altered upon binding the Mediator kinase module, a multi-subunit complex that contains CDK8 or its vertebrate-specific paralog CDK19. Here, we review the mechanisms by which the Mediator kinase module controls pol II transcription, emphasizing its impact on TF activity, pol II elongation, enhancer function, and chromatin architecture. We also highlight how the Mediator kinase module integrates signaling pathways with transcription to enable rapid, stimulus-specific responses, as well as its links to human disease.

Systematic Gene-to-Phenotype Arrays: A High-Throughput Technique for Molecular Phenotyping.

We have developed a highly parallel strategy, systematic gene-to-phenotype arrays (SGPAs), to comprehensively map the genetic landscape driving molecular phenotypes of interest. By this approach, a complete yeast genetic mutant array is crossed with fluorescent reporters and imaged on membranes at high density and contrast. Importantly, SGPA enables quantification of phenotypes that are not readily detectable in ordinary genetic analysis of cell fitness. We benchmark SGPA by examining two fundamental biological phenotypes: first, we explore glucose repression, in which SGPA identifies a requirement for the Mediator complex and a role for the CDK8/kinase module in regulating transcription. Second, we examine selective protein quality control, in which SGPA identifies most known quality control factors along with U34 tRNA modification, which acts independently of proteasomal degradation to limit misfolded protein production. Integration of SGPA with other fluorescent readouts will enable genetic dissection of a wide range of biological pathways and conditions.

Mediator kinase subunit CDK8 phosphorylates transcription factor TCP15 during tomato pollen development.

Pollen development in flowering plants has strong implications for reproductive success. Pollen DNA can be targeted to improve plant traits for yield and stress tolerance. In this study, we demonstrated that the Mediator subunit CYCLIN-DEPENDENT KINASE 8 (CDK8) is a key modulator of pollen development in tomato (Solanum lycopersicum). SlCDK8 knockout led to significant decreases in pollen viability, fruit yield, and fruit seed number. We also found that SlCDK8 directly interacts with transcription factor TEOSINTE BRANCHED1-CYCLOIDEA-PCF15 (SlTCP15) using yeast two-hybrid screens. We subsequently showed that SlCDK8 phosphorylates Ser 187 of SlTCP15 to promote SlTCP15 stability. Phosphorylated TCP15 directly bound to the TGGGCY sequence in the promoters of DYSFUNCTIONAL TAPETUM 1 (SlDYT1) and MYB DOMAIN PROTEIN 103 (SlMYB103), which are responsible for pollen development. Consistently, disruption of SlTCP15 resembled slcdk8 tomato mutants. In sum, our work identified a new substrate of Mediator CDK8 and revealed an important regulatory role of SlCDK8 in pollen development via cooperation with SlTCP15.

MPK1/SLT2 Links Multiple Stress Responses with Gene Expression in Budding Yeast by Phosphorylating Tyr1 of the RNAP II CTD.

The RNA polymerase II largest subunit C-terminal domain consists of repeated YSPTSPS heptapeptides. The role of tyrosine-1 (Tyr1) remains incompletely understood, as, for example, mutating all Tyr1 residues to Phe (Y1F) is lethal in vertebrates but a related mutant has only a mild phenotype in S. pombe. Here we show that Y1F substitution in budding yeast resulted in a strong slow-growth phenotype. The Y1F strain was also hypersensitive to several different cellular stresses that involve MAP kinase signaling. These phenotypes were all linked to transcriptional changes, and we also identified genetic and biochemical interactions between Tyr1 and both transcription initiation and termination factors. Further studies uncovered defects related to MAP kinase I (Slt2) pathways, and we provide evidence that Slt2 phosphorylates Tyr1 in vitro and in vivo. Our study has thus identified Slt2 as a Tyr1 kinase, and in doing so provided links between stress response activation and Tyr1 phosphorylation.

Inhibition of CDK8/19 Mediator kinase potentiates HER2-targeting drugs and bypasses resistance to these agents in vitro and in vivo.

Breast cancers (BrCas) that overexpress oncogenic tyrosine kinase receptor HER2 are treated with HER2-targeting antibodies (such as trastuzumab) or small-molecule kinase inhibitors (such as lapatinib). However, most patients with metastatic HER2+ BrCa have intrinsic resistance and nearly all eventually become resistant to HER2-targeting therapy. Resistance to HER2-targeting drugs frequently involves transcriptional reprogramming associated with constitutive activation of different signaling pathways. We have investigated the role of CDK8/19 Mediator kinase, a regulator of transcriptional reprogramming, in the response of HER2+ BrCa to HER2-targeting drugs. CDK8 was in the top 1% of all genes ranked by correlation with shorter relapse-free survival among treated HER2+ BrCa patients. Selective CDK8/19 inhibitors (senexin B and SNX631) showed synergistic interactions with lapatinib and trastuzumab in a panel of HER2+ BrCa cell lines, overcoming and preventing resistance to HER2-targeting drugs. The synergistic effects were mediated in part through the PI3K/AKT/mTOR pathway and reduced by PI3K inhibition. Combination of HER2- and CDK8/19-targeting agents inhibited STAT1 and STAT3 phosphorylation at S727 and up-regulated tumor suppressor BTG2. The growth of xenograft tumors formed by lapatinib-sensitive or -resistant HER2+ breast cancer cells was partially inhibited by SNX631 alone and strongly suppressed by the combination of SNX631 and lapatinib, overcoming lapatinib resistance. These effects were associated with decreased tumor cell proliferation and altered recruitment of stromal components to the xenograft tumors. These results suggest potential clinical benefit of combining HER2- and CDK8/19-targeting drugs in the treatment of metastatic HER2+ BrCa.

The Candida albicans Cdk8-dependent phosphoproteome reveals repression of hyphal growth through a Flo8-dependent pathway.

Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent transcriptional and morphological changes, suggesting that Ssn3 kinase activity negatively regulates Flo8.Under embedded conditions, when ssn3Δ/Δ and efg1Δ/Δ mutants were hyperfilamentous, FLO8 was essential for hypha formation. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that the ssn3Δ/Δ medium alkalinization phenotype, which is dependent on STP2, a transcription factor involved in amino acid utilization, also requires FLO8 and EFG1. Together, these data show that Ssn3 activity can modulate Flo8 and its direct and indirect interactions in different ways, and underscores the potential importance of considering Ssn3 function in the control of transcription factor activities.

Cbx3 maintains lineage specificity during neural differentiation.

Chromobox homolog 3 (Cbx3/heterochromatin protein 1γ [HP1γ]) stimulates cell differentiation, but its mechanism is unknown. We found that Cbx3 binds to gene promoters upon differentiation of murine embryonic stem cells (ESCs) to neural progenitor cells (NPCs) and recruits the Mediator subunit Med26. RNAi knockdown of either Cbx3 or Med26 inhibits neural differentiation while up-regulating genes involved in mesodermal lineage decisions. Thus, Cbx3 and Med26 together ensure the fidelity of lineage specification by enhancing the expression of neural genes and down-regulating genes specific to alternative fates.

Expanding the phenotypic spectrum for CDK8-related disease: A case report.

BACKGROUND: Cyclin-dependent kinase 8 (CDK8) is part of a regulatory kinase module that regulates the activity of the Mediator complex. The Mediator, a large conformationally flexible protein complex, goes on to regulate RNA polymerase II activity, consequently affecting transcriptional regulation. Thus, inactivating mutations of the genes within the kinase module cause aberrant transcriptional regulation and disease, namely, CDK8-related intellectual developmental disorder with hypotonia and behavioral abnormalities (IDDHBA). CASE PRESENTATION: We describe, for the first time, a likely pathogenic heterozygous CDK8 variant c.599G>A, p.(Arg200Gln) inherited from the biological mother. The clinical presentation of the child and mother is within the described clinical spectrum for IDDHBA; however, undocumented progressive contractures of the hips and knees as well as scoliosis were also observed in the child. This phenotype was not found in the mother, highlighting a heterogenous presentation for the same variant within the same family. Furthermore, the described clinical presentation may further support the notion of a module- or Mediator-related syndrome with varying clinical presentation. CONCLUSION: This case report documents the first inherited case of IDDHBA and expands the phenotypic spectrum for CDK8-related disease to include undocumented progressive contractures of the hips and knees as well as scoliosis, which may support the notion of a module- or Mediator-related syndrome with varying clinical presentation.

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. We have 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 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.

Osteoblastgenic and Osteogenic Effects of KY-273 with CDK8/19 Inhibitory Activity in Bone Marrow Mesenchymal Stem Cells and Female Rats.

Osteoporosis is caused by imbalance between osteogenesis and bone resorption, thus, osteogenic drugs and resorption inhibitors are used for treatment of osteoporosis. The present study examined the effects of (R)-4-(1-hydroxyethyl)-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzamide (KY-273), a diphenyl ether derivative, on CDK8/19 activity, osteoblast differentiation and femoral bone using micro-computed tomography in female rats. KY-273 potently inhibited CDK8/19 activity, promoted osteoblast differentiation with an increase in alkaline phosphatase (ALP) activity, and gene expression of type I collagen, ALP and BMP-4 in mesenchymal stem cells (ST2 cells). In female rat femur, ovariectomy decreased metaphyseal trabecular bone volume (Tb.BV), mineral content (Tb.BMC), yet had no effect on metaphyseal and diaphyseal cortical bone volume (Ct.BV), mineral content (Ct.BMC) and strength parameters (BSPs). In ovaries-intact and ovariectomized rats, oral administration of KY-273 (10 mg/kg/d) for 6 weeks increased metaphyseal and diaphyseal Ct.BV, Ct.BMC, and BSPs without affecting medullary volume (Med.V), but did not affect Tb.BV and Tb.BMC. In ovariectomized rats, alendronate (3 mg/kg/d) caused marked restoration of Tb.BV, Tb.BMC and structural parameters after ovariectomy, and increased metaphyseal but not diaphyseal Ct.BV, Ct.BMC, and BSPs. In ovaries-intact and ovariectomized rats, by the last week, KY-273 increased bone formation rate/bone surface at the periosteal but not the endocortical side. These findings indicate that KY-273 causes osteogenesis in cortical bone at the periosteal side without reducing Med.V. In conclusion, KY-273 has cortical-bone-selective osteogenic effects by osteoblastogenesis via CDK8/19 inhibition in ovaries-intact and ovariectomized rats, and is an orally active drug candidate for bone diseases such as osteoporosis in monotherapy and combination therapy.

Circular RNA MKLN1 promotes epithelial-mesenchymal transition in pulmonary fibrosis by regulating the miR-26a/b-5p/CDK8 axis in human alveolar epithelial cells and mice models.

Pulmonary fibrosis involves destruction of the lung parenchyma and extracellular matrix deposition. Effective treatments for pulmonary fibrosis are lacking and its pathogenesis is still unclear. Studies have found that epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AECs) plays an important role in progression of pulmonary fibrosis. Thus, an in-depth exploration of its mechanism might identify new therapeutic targets. In this study, we revealed that a novel circular RNA, MKLN1 (circMKLN1), was significantly elevated in two pulmonary fibrosis models (intraperitoneally with PQ, 50 mg/kg for 7 days, and intratracheally with BLM, 5 mg/kg for 28 days). Additionally, circMKLN1 was positively correlated with the severity of pulmonary fibrosis. Inhibition of circMKLN1 expression significantly reduced collagen deposition and inhibited EMT in AECs. EMT was aggravated after circMKLN1 overexpression in AECs. MiR-26a-5p/miR-26b-5p (miR-26a/b), the targets of circMKLN1, were confirmed by luciferase reporter assays. CircMKLN1 inhibition elevated miR-26a/b expression. Significantly decreased expression of CDK8 (one of the miR-26a/b targets) was observed after inhibition of circMKLN1. EMT was exacerbated again, and CDK8 expression was significantly increased after circMKLN1 inhibition and cotransfection of miR-26a/b inhibitors in AECs. Our research indicated that circMKLN1 promoted CDK8 expression through sponge adsorption of miR-26a/b, which regulates EMT and pulmonary fibrosis. This study provides a theoretical basis for finding new targets or biomarkers in pulmonary fibrosis.

Discovery of novel and potent CDK8 inhibitors for the treatment of acute myeloid leukaemia.

It has been reported that CDK8 plays a key role in acute myeloid leukaemia. Here, a total of 40 compounds were rational designed and synthesised based on the previous SAR. Among them, compound 12 (3-(3-(furan-3-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide) showed the most potent inhibiting activity against CDK8 with an IC50 value of 39.2 ± 6.3 nM and anti AML cell proliferation activity (molm-13 GC50 = 0.02 ± 0.01 µM, MV4-11 GC50 = 0.03 ± 0.01 µM). Mechanistic studies revealed that this compound 12 could inhibit the phosphorylation of STAT-1 and STAT-5. Importantly, compound 12 showed relative good bioavailability (F = 38.80%) and low toxicity in vivo. This study has great significance for the discovery of more efficient CDK8 inhibitors and the development of drugs for treating AML in the future.

Highlighting the Major Role of Cyclin C in Cyclin-Dependent Kinase 8 Activity through Molecular Dynamics Simulations.

Dysregulation of cyclin-dependent kinase 8 (CDK8) activity has been associated with many diseases, including colorectal and breast cancer. As usual in the CDK family, the activity of CDK8 is controlled by a regulatory protein called cyclin C (CycC). But, while human CDK family members are generally activated in two steps, that is, the binding of the cyclin to CDK and the phosphorylation of a residue in the CDK activation loop, CDK8 does not require the phosphorylation step to be active. Another peculiarity of CDK8 is its ability to be associated with CycC while adopting an inactive form. These specificities raise the question of the role of CycC in the complex CDK8-CycC, which appears to be more complex than the other members of the CDK family. Through molecular dynamics (MD) simulations and binding free energy calculations, we investigated the effect of CycC on the structure and dynamics of CDK8. In a second step, we particularly focused our investigation on the structural and molecular basis of the protein-protein interaction between the two partners by finely analyzing the energetic contribution of residues and simulating the transition between the active and the inactive form. We found that CycC has a stabilizing effect on CDK8, and we identified specific interaction hotspots within its interaction surface compared to other human CDK/Cyc pairs. Targeting these specific interaction hotspots could be a promising approach in terms of specificity to effectively disrupt the interaction between CDK8. The simulation of the conformational transition from the inactive to the active form of CDK8 suggests that the residue Glu99 of CycC is involved in the orientation of three conserved arginines of CDK8. Thus, this residue may assume the role of the missing phosphorylation step in the activation mechanism of CDK8. In a more general view, these results point to the importance of keeping the CycC in computational studies when studying the human CDK8 protein in both the active and the inactive form.

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

We identified three unrelated individuals 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, our 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.

Cdk8 is required for establishment of H3K27me3 and gene repression by Xist and mouse development.

We previously identified the cyclin dependent kinase Cdk8 as a putative silencing factor for Xist To investigate its role in X inactivation, we engineered a Cdk8 mutation in mouse embryonic stem cells (ESCs) carrying an inducible system for studying Xist function. We found that Xist repressed X-linked genes at half of the expression level in Cdk8 mutant cells, whereas they were almost completely silenced in the controls. Lack of Cdk8 impaired Ezh2 recruitment and the establishment of histone H3 lysine 27 tri-methylation but not PRC1 recruitment by Xist Transgenic expression of wild-type but not catalytically inactive Cdk8 restored efficient gene repression and PRC2 recruitment. Mutation of the paralogous kinase Cdk19 did not affect Xist function, and combined mutations of Cdk8 and Cdk19 resembled the Cdk8 mutation. In mice, a Cdk8 mutation caused post-implantation lethality. We observed that homozygous Cdk8 mutant female embryos showed a greater developmental delay than males on day 10.5. Together with the inefficient repression of X-linked genes in differentiating Cdk8 mutant female ESCs, these data show a requirement for Cdk8 in the initiation of X inactivation.