Abstract
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.