ABSTRACT
The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs.
Subject(s)
CDC2 Protein Kinase/metabolism , Embryonic Stem Cells/physiology , Epigenesis, Genetic , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Animals , CDC2 Protein Kinase/genetics , Cell Differentiation , Cells, Cultured , Chromatin Immunoprecipitation/methods , Female , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , MCF-7 Cells , Male , Mice , Mice, Knockout , Phosphorylation , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
BACKGROUND: The effort to characterize intrinsically disordered regions of signaling proteins is rapidly expanding. An important class of disordered interaction modules are ubiquitous and functionally diverse elements known as short linear motifs (SLiMs). RESULTS: To further examine the role of SLiMs in signal transduction, we used a previously devised bioinformatics method to predict evolutionarily conserved SLiMs within a well-characterized pathway in S. cerevisiae. Using a single cell, reporter-based flow cytometry assay in conjunction with a fluorescent reporter driven by a pathway-specific promoter, we quantitatively assessed pathway output via systematic deletions of individual motifs. We found that, when deleted, 34% (10/29) of predicted SLiMs displayed a significant decrease in pathway output, providing evidence that these motifs play a role in signal transduction. Assuming that mutations in SLiMs have quantitative effects on mechanisms of signaling, we show that perturbations of parameters in a previously published stochastic model of HOG signaling could reproduce the quantitative effects of 4 out of 7 mutations in previously unknown SLiMs. CONCLUSIONS: Our study suggests that, even in well-characterized pathways, large numbers of functional elements remain undiscovered, and that challenges remain for application of systems biology models to interpret the effects of mutations in signaling pathways.
