A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation

A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation

A molecular mechanism for the quantitativemodel of the cell cycle : CDK-mediatedphosphorylation of intrinsically disordered regions

La progression du cycle cellulaire eucaryote est contrôlée par la famille des kinases dépendantes des cyclines (CDK). Ces enzymes, en complexe avec les cyclines, phosphorylent des substrats pour progresser dans les différentes phases du cycle cellulaire. À l'opposé de ce modèle qualitatif, le "modèle quantitatif" propose que la plupart des fonctions des CDK-cyclines sont redondantes et que ce sont les niveaux d'activité globale des CDK qui pilotent le cycle cellulaire. Les systèmes génétiques qui permettent aux scientifiques de manipuler les niveaux individuels de CDK sont essentiels pour répondre aux questions critiques qui aident à élucider quel modèle explique le mieux l'ensemble des preuves actuelles. Je présente ici mes avancées dans la conception de tels systèmes à introduire dans des cellules ayant des contextes génétiques différents.L'un des principaux dilemmes à résoudre en considérant le modèle quantitatif est de savoir comment une activité globale peut contrôler précisément le cycle cellulaire. Je suggère ici un mécanisme d'action par lequel la phosphorylation de régions intrinsèquement désordonnées des protéines contrôle la formation et la dissolution de condensats protéiques agissant comme des plaques tournantes biochimiques dans la cellule. CDK et des autres kinases du cycle cellulaire partagent la tendance à phosphoryler les régions désordonnées et leurs substrats contiennent plus de ces régions que le reste des protéines phosphorylées. De plus, une proportion frappante de protéines dans les condensats protéiques sont des cibles des CDK.Nous avons obtenu une carte de phosphorylation à haute résolution à travers la première division cellulaire d'embryons uniques de Xenopus laevis et avons confirmé qu'avant la mitose, une augmentation rapide de la phosphorylation globale se produit, la plupart étant médiée par la CDK. Nous avons détecté un nombre élevé de phosphorylations en interphase qui étaient enrichies en motifs de phosphorylation CDK et autres kinases du cycle cellulaire.Nous avons choisi le marqueur de prolifération Ki-67 comme étude de cas pour examiner comment la phosphorylation peut réguler le processus de séparation des phases, responsable de la formation des condensats moléculaires. Ki67 semble présenter des modes concurrents de régulation de la séparation de phase par la phosphorylation de son domaine répété, qui dépendent du contexte cellulaire et moléculaire. Il semble que différents niveaux de phosphorylation localiseront de manière différentielle le Ki-67 à l'hétérochromatine périnucléolaire pendant l'interphase et à la couche périchromosomique en mitose, toutes deux décrites comme étant en phase séparée.La théorie de l'action globale de la phosphorylation de la CDK dans le contrôle de la formation des centres biochimiques où se produisent les réactions spécifiques du cycle cellulaire manque cependant d'une explication détaillée des effets en aval de cette régulation. En prenant Ki-67 comme effecteur de la régulation de la séparation des phases médiée par les CDK, nous avons étudié les modifications de l'organisation de la chromatine dans les cellules dépourvues de Ki-67. Nous avons constaté que le knock-out de Ki-67 produit des changements massifs dans le transcriptome, partiellement dus à des changements dans les marques d'histones de la chromatine. Un autre mécanisme d'action de la phosphorylation des régions désordonnées des protéines médiée par les CDK pourrait être la régulation de l'activité des condensats auxquels ils participent activement. Les membres de la sous-famille CDK8/19 en complexe avec la cycline C constituent le domaine kinase du complexe Mediator, qui phosphoryle le domaine C-terminal désordonné de Pol II. La délétion à la fois de CDK8 et de CDK19 produit des altérations transcriptionnelles dans de multiples gènes, bien que ces changements soient plutôt de faible amplitude et que les changements transcriptionnels observés semblent dépendre du contexte cellulaire.Eukaryotic cell cycle progression is controlled by Cyclin-Dependent Kinase family (CDK). Theseenzymes in complex with their mandatory binding partners, the cyclins, phosphorylate substrates to progress through the different phases of the cell cycle. Opposed to the qualitative model, an alternative model proposes that most CDK-cyclin function is redundant and it is the global CDK activity levels that drive the cell cycle. This so-called “quantitative model”, implies that there exist low and high overall CDK activity thresholds for entry into S-phase and mitosis, respectively, determined by the CDK regulatory network. Genetic systems that allow scientists to manipulate individual CDK levels are pivotal to addressing critical questions that help to elucidate which model explains better the current body of evidence.Here I present my advances in the design of such systems to be introduced into cells with different genetic contexts and into model organisms, as well. One of the main dilemmas to be solved when considering the quantitative model is how a global activity can precisely control all the biochemical states of the cell during the cell cycle. Here, I suggest a mechanism of action by which phosphorylation of intrinsically disordered regions of proteins control the formation and dissolution of protein condensates acting as biochemical hubs in the cell. Not only CDKs but most cell cycle kinases share the tendency of phosphorylating disordered regions and their substrates contain more of these regions that the rest of the phosphorylated proteins. Moreover, a striking proportion of proteins in the protein condensates are CDK targets.We obtained a high- resolution phosphorylation map through the first cell division of single embryos of Xenopus laevis and confirmed that before mitosis, a rapid increase of global phosphorylation occurs, most being CDK-mediated. We detected a high number of interphase phosphorylations that were enriched in CDK phosphorylation motifs and other cell cycle kinases.We selected the proliferation marker Ki-67 as a case study to investigate how phosphorylation can regulate the process of phase separation, responsible for the formation of the molecular condensates. Ki67 appears to present competing modes of regulation of phase separation by phosphorylation of its repeat domain, which will depend on the cellular and molecular context. It appears that different levels of phosphorylation will differentially localize Ki-67 to the perinucleolar heterochromatin during interphase and to the perichromosomal layer in mitosis, both described as being phase-separated.The theory of global action of the CDK phosphorylation in controlling the formation of biochemical centers where cell cycle-specific reaction happens lacks, however, a detailed explanation of the downstream effects of this regulation. Taking Ki-67 as an effector of CDK-mediated regulation of phase separation, we investigated what are the changes in chromatin organization in cells lacking Ki-67. We found that knockout of Ki-67 produces massive changes in the transcriptome, partially due to changes in chromatin histone marks, especially the inhibitory H3K27 trimethylation. Another mechanism of action of CDK-mediated phosphorylation of disordered regions of proteins might be the regulation of the activity of condensates in which they participate actively. Members of the CDK8/19 subfamily in complex with cyclin C constitute the kinase domain of the Mediator complex, which phosphorylates the disordered C-terminal domain of Pol II. Several other subunits of the Mediator are also reported to be disordered and this complex appears to undergo phase separation and form the so-called “super-enhancers”. The deletion of both CDK8 and CDK19 produces transcriptional alterations in multiple genes, although those changes are rather low in magnitude and the transcriptional changes observed seem to be dependent on the cellular context

Un mécanisme moléculaire pour le modèlequantitatif du cycle cellulaire : laphosphorylation par les CDK des régionsintrinsèquement non structurées.

Eukaryotic cell cycle progression is controlled by Cyclin-Dependent Kinase family (CDK). Theseenzymes in complex with their mandatory binding partners, the cyclins, phosphorylate substrates to progress through the different phases of the cell cycle. Opposed to the qualitative model, an alternative model proposes that most CDK-cyclin function is redundant and it is the global CDK activity levels that drive the cell cycle. This so-called “quantitative model”, implies that there exist low and high overall CDK activity thresholds for entry into S-phase and mitosis, respectively, determined by the CDK regulatory network. Genetic systems that allow scientists to manipulate individual CDK levels are pivotal to addressing critical questions that help to elucidate which model explains better the current body of evidence.Here I present my advances in the design of such systems to be introduced into cells with different genetic contexts and into model organisms, as well. One of the main dilemmas to be solved when considering the quantitative model is how a global activity can precisely control all the biochemical states of the cell during the cell cycle. Here, I suggest a mechanism of action by which phosphorylation of intrinsically disordered regions of proteins control the formation and dissolution of protein condensates acting as biochemical hubs in the cell. Not only CDKs but most cell cycle kinases share the tendency of phosphorylating disordered regions and their substrates contain more of these regions that the rest of the phosphorylated proteins. Moreover, a striking proportion of proteins in the protein condensates are CDK targets.We obtained a high- resolution phosphorylation map through the first cell division of single embryos of Xenopus laevis and confirmed that before mitosis, a rapid increase of global phosphorylation occurs, most being CDK-mediated. We detected a high number of interphase phosphorylations that were enriched in CDK phosphorylation motifs and other cell cycle kinases.We selected the proliferation marker Ki-67 as a case study to investigate how phosphorylation can regulate the process of phase separation, responsible for the formation of the molecular condensates. Ki67 appears to present competing modes of regulation of phase separation by phosphorylation of its repeat domain, which will depend on the cellular and molecular context. It appears that different levels of phosphorylation will differentially localize Ki-67 to the perinucleolar heterochromatin during interphase and to the perichromosomal layer in mitosis, both described as being phase-separated.The theory of global action of the CDK phosphorylation in controlling the formation of biochemical centers where cell cycle-specific reaction happens lacks, however, a detailed explanation of the downstream effects of this regulation. Taking Ki-67 as an effector of CDK-mediated regulation of phase separation, we investigated what are the changes in chromatin organization in cells lacking Ki-67. We found that knockout of Ki-67 produces massive changes in the transcriptome, partially due to changes in chromatin histone marks, especially the inhibitory H3K27 trimethylation. Another mechanism of action of CDK-mediated phosphorylation of disordered regions of proteins might be the regulation of the activity of condensates in which they participate actively. Members of the CDK8/19 subfamily in complex with cyclin C constitute the kinase domain of the Mediator complex, which phosphorylates the disordered C-terminal domain of Pol II. Several other subunits of the Mediator are also reported to be disordered and this complex appears to undergo phase separation and form the so-called “super-enhancers”. The deletion of both CDK8 and CDK19 produces transcriptional alterations in multiple genes, although those changes are rather low in magnitude and the transcriptional changes observed seem to be dependent on the cellular context.La progression du cycle cellulaire eucaryote est contrôlée par la famille des kinases dépendantes des cyclines (CDK). Ces enzymes, en complexe avec les cyclines, phosphorylent des substrats pour progresser dans les différentes phases du cycle cellulaire. À l'opposé de ce modèle qualitatif, le "modèle quantitatif" propose que la plupart des fonctions des CDK-cyclines sont redondantes et que ce sont les niveaux d'activité globale des CDK qui pilotent le cycle cellulaire. Les systèmes génétiques qui permettent aux scientifiques de manipuler les niveaux individuels de CDK sont essentiels pour répondre aux questions critiques qui aident à élucider quel modèle explique le mieux l'ensemble des preuves actuelles. Je présente ici mes avancées dans la conception de tels systèmes à introduire dans des cellules ayant des contextes génétiques différents.L'un des principaux dilemmes à résoudre en considérant le modèle quantitatif est de savoir comment une activité globale peut contrôler précisément le cycle cellulaire. Je suggère ici un mécanisme d'action par lequel la phosphorylation de régions intrinsèquement désordonnées des protéines contrôle la formation et la dissolution de condensats protéiques agissant comme des plaques tournantes biochimiques dans la cellule. CDK et des autres kinases du cycle cellulaire partagent la tendance à phosphoryler les régions désordonnées et leurs substrats contiennent plus de ces régions que le reste des protéines phosphorylées. De plus, une proportion frappante de protéines dans les condensats protéiques sont des cibles des CDK.Nous avons obtenu une carte de phosphorylation à haute résolution à travers la première division cellulaire d'embryons uniques de Xenopus laevis et avons confirmé qu'avant la mitose, une augmentation rapide de la phosphorylation globale se produit, la plupart étant médiée par la CDK. Nous avons détecté un nombre élevé de phosphorylations en interphase qui étaient enrichies en motifs de phosphorylation CDK et autres kinases du cycle cellulaire.Nous avons choisi le marqueur de prolifération Ki-67 comme étude de cas pour examiner comment la phosphorylation peut réguler le processus de séparation des phases, responsable de la formation des condensats moléculaires. Ki67 semble présenter des modes concurrents de régulation de la séparation de phase par la phosphorylation de son domaine répété, qui dépendent du contexte cellulaire et moléculaire. Il semble que différents niveaux de phosphorylation localiseront de manière différentielle le Ki-67 à l'hétérochromatine périnucléolaire pendant l'interphase et à la couche périchromosomique en mitose, toutes deux décrites comme étant en phase séparée.La théorie de l'action globale de la phosphorylation de la CDK dans le contrôle de la formation des centres biochimiques où se produisent les réactions spécifiques du cycle cellulaire manque cependant d'une explication détaillée des effets en aval de cette régulation. En prenant Ki-67 comme effecteur de la régulation de la séparation des phases médiée par les CDK, nous avons étudié les modifications de l'organisation de la chromatine dans les cellules dépourvues de Ki-67. Nous avons constaté que le knock-out de Ki-67 produit des changements massifs dans le transcriptome, partiellement dus à des changements dans les marques d'histones de la chromatine. Un autre mécanisme d'action de la phosphorylation des régions désordonnées des protéines médiée par les CDK pourrait être la régulation de l'activité des condensats auxquels ils participent activement. Les membres de la sous-famille CDK8/19 en complexe avec la cycline C constituent le domaine kinase du complexe Mediator, qui phosphoryle le domaine C-terminal désordonné de Pol II. La délétion à la fois de CDK8 et de CDK19 produit des altérations transcriptionnelles dans de multiples gènes, bien que ces changements soient plutôt de faible amplitude et que les changements transcriptionnels observés semblent dépendre du contexte cellulaire

Tubulin glutamylation: a skeleton key for neurodegenerative diseases

International audiencePeRSPeCTIve Tubulin glutamylation: a skeleton key for neurodegenerative diseases Microtubules (MTs) are cytoskeletal elements formed by a non-cova-lent association of α-and β-tubulin heterodimers. They provide structure and shape to all eukaryotic cells and are implicated in a variety of fundamental cellular processes including cell motility, cell division, mechanotransduction as well as long-distance intracellular cargo transport. In neurons, they constitute the molecular frame that maintains the lengthy axonal projections. In view of the relative size of some ax-ons in the human body, which can reach up to 1 m, the active transport of e.g., vesicles over the MT arrays to the synaptic cleft, is of particular importance. Considering the numerous roles of MTs, it is not surprising that already 30 years ago, impairment of the MT-based system was proposed as a unifying hypothesis for the variable clinical presentations in Alzheimer's disease (Matsuyama and Jarvik, 1989). In this context, a key question is how the MT network accommodates all these different functions, often within the same cell? Current view is that every MT-dependent process is executed through the recruitment of a specific set of MT-associated proteins (MAPs) and molecular motors. Thus, it is of fundamental importance to understand how recruitment of these MAPs and motors is regulated. Since many of the MAPs and motors bind to the C-terminal tails of α-and β-tubulin, which are known to protrude from the MT surface, one important mechanism by which MTs may regulate the association of the effector proteins is through posttranslational modifications (PTMs). The modifications that occur on the C-terminal tails consists of either addition or removal of amino acids including polyglutamylation, polyglycylation and detyrosination. Very recently we have identified the members of the vasohibin family as cysteine proteases responsible for tubulin detyrosination (Aillaud et al., 2017), a modification, which consists of proteolytic removal of the very C-terminal tyrosine residue present on α-tubulin. The reverse reaction that consists of reattachment of a tyrosine residue is carried out by an enzyme called tubulin tyrosine ligase (TTL). Moreover, the C-terminal tails of both α-and β-tubulin are also subjected to polymodifications namely polyglutamylation and polyglycylation. These modifications are reversible and consist of the enzymatic addition of sidechains composed of either glutamate or glycine to the gamma carboxyl groups of primary sequence gluta-mates. The enzymes, involved in the addition of both glutamylation and glycylation side chains, share a homology domain with TTL and thus are called tubulin tyrosine ligase like (TTLL). The human genome contains thirteen TTLL related genes. Nine of them are involved in tubulin polyglutamylation (TTLL1, TTLL2, TTLL4, TTLL5, TTLL6, TTLL7, TTLL9, TTLL11 and TTLL13) and three in tubulin polyglyc-ylation (TTLL3, TTLL8 and TTLL10) (Rogowski et al., 2009), while one, TTLL12, remains without assigned function. On the other hand, tubulin deglutamylation has been shown to be catalyzed by a family of cytosolic carboxypeptidases (CCP), which is composed of six members (Rogowski et al., 2010). In contrast, the enzymes responsible for de-glycylation remain to be discovered. Overall, this complex enzymatic machinery allows for spatial and temporal fine-tuning of the physico-chemical properties of the MTs surface, ensuring functional diversification. In analogy to the "histone code", this regulatory system was originally coined as the "tubulin code" in a seminal review (Verhey and Gaertig, 2007). A proof of concept of the "tubulin code" was provided in the context of in vitro studies showing that PTMs confer unique biochemical properties, drive dynein and kinesin motor velocity, proces-sivity and the rates of MT depolymerisation (Sirajuddin et al., 2014). While polyglycylation appears to be specific to cilia and flagella, polyglutamylation and detyrosination are more ubiquitous. Biochemical characterization of MTs obtained from brain tissue revealed the presence of extensive PTMs on the protruding C-terminal tails of α-and β-tubulin with the most abundant modification being polyglutam-ylation. The first enzyme involved in glutamylation to be identified, TTLL1, was originally purified from mouse brain using classical biochemistry , and confirmed genetically by developing knockout Tetra-hymena cells, which lacked homologous gene and showed reduced level of glutamylation (Janke et al., 2005). A comprehensive follow up study demonstrated that in humans, apart from TTLL1, eight additional members of the TTLL family encode tubulin glutamylases. These enzymes are characterized by different specificities with some of them preferentially being involved in initiation while the others in the elongation of the glutamate chain. The identification of the reverse enzymes, the CCPs, came with the analysis of Purkinje cell degenera-tion (pcd) mouse model. These mice exhibit ataxia, which results from postnatal degeneration of almost all Purkinje cells in the cerebellum. Genetic analysis revealed that pcd mice carry a mutation in the CCP1 gene, which encodes a protein having tubulin deglutamylase activity. As such, pcd mice display abnormally high level of polyglutamylation in the cerebellar neurons. Stunningly, the Purkinje cell degeneration phenotype observed in the pcd mice was rescued by a knockdown of TTLL1 glutamylase, demonstrating that neuronal death is indeed mediated by tubulin hyperglutamylation. These observations provided the first molecular link between altered levels of tubulin glutamylation and neurodegeneration (Rogowski et al., 2010). Current view limits the regulation of tubulin glutamylation levels in the cells to direct competition between the forward and reverse enzymes and does not include additional regulators. Recently, we have identified cilia and spindle-associated protein (CSAP) as a master regulator of tubulin glutamylases (Bompard et al., 2018). We found that expression of CSAP enhances overall activity of all autonomously active glutamylating enzymes and in the case of TTLL5 and TTLL7 also potentiates their elongase activity. Moreover, biochemical analysis revealed that CSAP interacts with TTLL glutamylases and appears to regulate their protein abundance through stabilization (Figure 1A). In turn, due to its high affinity for MTs, CSAP redirects glutamylase activity from tubulin towards MTs. By exploring the human protein atlas (Uhlén et al., 2015), we found that CSAP has a striking distribution in human tissue and is preferentially expressed in brain (Figure 1B). Thus, we propose that neurons utilize the expression of CSAP, as a regulatory Figure 1 Cilia and spindle-associated protein (CSAP) function and tissue distribution in humans. (A) Schematic representation of the role of CSAP protein in glutamylation of microtubules. (B) Protein expression of CSAP in different organs of the human body. Data are obtained from Human Protein Atlas available from www.proteinatlas.org. TTLL: Tubulin tyrosine ligase like; CCP: cyto-solic carboxypeptidases. A B [Downloaded free from http://www.nrronline.org on Thursday, December 5, 2019, IP: 195.83.84.168

CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium

International audienceCDK8 and CDK19 form a conserved cyclin-dependent kinase subfamily that interacts with the essential transcription complex, Mediator, and also phosphorylates the C-terminal domain of RNA polymerase II. Cells lacking either CDK8 or CDK19 are viable and have limited transcriptional alterations, but whether the two kinases redundantly control cell proliferation and differentiation is unknown. Here, we find in mice that CDK8 is dispensable for regulation of gene expression, normal intestinal homeostasis, and efficient tumourigenesis, and is largely redundant with CDK19 in the control of gene expression. Their combined deletion in intestinal organoids reduces long-term proliferative capacity but is not lethal and allows differentiation. However, double-mutant organoids show mucus accumulation and increased secretion by goblet cells, as well as downregulation of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and functionality of the CFTR pathway. Pharmacological inhibition of CDK8/19 kinase activity in organoids and in mice recapitulates several of these phenotypes. Thus, the Mediator kinases are not essential for cell proliferation and differentiation in an adult tissue, but they cooperate to regulate specific transcriptional programmes

CDK8 and CDK19 kinases have non-redundant oncogenic functions in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common cancer with high mortality. The limited therapeutic options for advanced disease include treatment with Sorafenib, a multi-kinase inhibitor whose targets include the Mediator kinase CDK8. Since CDK8 has reported oncogenic activity in Wnt-dependent colorectal cancer, we investigated whether it is also involved in HCC. We find that CDK8 and its paralogue CDK19 are significantly overexpressed in HCC patients, where high levels correlate with poor prognosis. Liver-specific genetic deletion of CDK8 in mice is well supported and protects against chemical carcinogenesis. Deletion of either CDK8 or CDK19 in hepatic precursors had little effect on gene expression in exponential cell growth but prevented oncogene-induced transformation. This phenotype was reversed by concomitant deletion of TP53. These data support important and non-redundant roles for mediator kinases in liver carcinogenesis, where they genetically interact with the TP53 tumor suppressor.Hepatocellular carcinoma (HCC) is a common cancer with high mortality. The limited therapeutic options for advanced disease include treatment with Sorafenib, a multi-kinase inhibitor whose targets include the Mediator kinase CDK8. Since CDK8 has reported oncogenic activity in Wnt-dependent colorectal cancer, we investigated whether it is also involved in HCC. We find that CDK8 and its paralogue CDK19 are significantly overexpressed in HCC patients, where high levels correlate with poor prognosis. Liver-specific genetic deletion of CDK8 in mice is well supported and protects against chemical carcinogenesis. Deletion of either CDK8 or CDK19 in hepatic precursors had little effect on gene expression in exponential cell growth but prevented oncogene-induced transformation. This phenotype was reversed by concomitant deletion of TP53. These data support important and non-redundant roles for mediator kinases in liver carcinogenesis, where they genetically interact with the TP53 tumor suppressor

CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium

Abstract CDK8 and CDK19 form a conserved cyclin-dependent kinase subfamily that interacts with the essential transcription complex, Mediator, and also promotes transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II. Cells lacking either CDK8 or CDK19 are viable and have limited transcriptional alterations, but whether the two kinases redundantly control cell differentiation is unknown. Here, we find that CDK8 is dispensable for RNA polII CTD phosphorylation, regulation of gene expression, normal intestinal homeostasis and efficient tumourigenesis in mice. Furthermore, CDK8 is largely redundant with CDK19 in the control of gene expression. Yet, while their combined deletion in intestinal organoids reduces long-term proliferative capacity, it is not lethal and allows differentiation. Nevertheless, in double mutant organoids, the cystic fibrosis transmembrane conductance regulator (CFTR) pathway is transcriptionally and functionally downregulated, leading to mucus accumulation and increased secretion by goblet cells. This phenotype can be recapitulated by pharmacological inhibition of CDK8/19 kinase activity. Thus, the Mediator kinases are not essential for cell proliferation and differentiation, but they cooperate to regulate tissue-specific transcriptional programmes

Evolutionary Divergence of Enzymatic Mechanisms for Tubulin Detyrosination

International audienceThe two related members of the vasohibin family, VASH1 and VASH2, encode human tubulin detyrosinases. Here we demonstrate that, in contrast to VASH1, which requires binding of small vasohibin binding protein (SVBP), VASH2 has autonomous tubulin detyrosinating activity. Moreover, we demonstrate that SVBP acts as a bona fide activator of both enzymes. Phylogenetic analysis of the vasohibin family revealed that regulatory diversification of VASH-mediated tubulin detyrosination coincided with early vertebrate evolution. Thus, as a model organism for functional analysis, we used Trypanosoma brucei (Tb), an evolutionarily early-branched eukaryote that possesses a single VASH and encodes a terminal tyrosine on both a- and b-tubulin tails, both subject to removal. Remarkably, although detyrosination levels are high in the flagellum, TbVASH knockout parasites did not present any noticeable flagellar abnormalities. In contrast, we observed reduced proliferation associated with profound morphological and mitotic defects, underscoring the importance of tubulin detyrosination in cell division

Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis

International audienceKi-67 is a nuclear protein that is expressed in all proliferating vertebrate cells. Here, we demonstrate that, although Ki-67 is not required for cell proliferation, its genetic ablation inhibits each step of tumor initiation, growth, and metastasis. Mice lacking Ki-67 are resistant to chemical or genetic induction of intestinal tumorigenesis. In established cancer cells, Ki-67 knockout causes global transcriptome remodeling that alters the epithelial–mesenchymal balance and suppresses stem cell characteristics. When grafted into mice, tumor growth is slowed, and metastasis is abrogated, despite normal cell proliferation rates. Yet, Ki-67 loss also down-regulates major histocompatibility complex class I antigen presentation and, in the 4T1 syngeneic model of mammary carcinoma, leads to an immune-suppressive environment that prevents the early phase of tumor regression. Finally, genes involved in xenobiotic metabolism are down-regulated, and cells are sensitized to various drug classes. Our results suggest that Ki-67 enables transcriptional programs required for cellular adaptation to the environment. This facilitates multiple steps of carcinogenesis and drug resistance, yet may render cancer cells more susceptible to antitumor immune responses