A systems-wide analysis of protein methylation in pluripotency

Abstract

Embryonic stem cells (ESCs) are pluripotent and can differentiate into all the cell types of an adult organism, hence they hold promise for regenerative medicine applications. Among other features, murine ESCs (mESCs) possess a highly permissive chromatin state, regulated by DNA methylation and post-translational modifications of histones, including methylation. Technological advances in mass spectrometry and the development of immune-purification techniques have revealed a landscape of biological functions regulated by protein methylation beyond histones. Indeed, multiple lines of evidence indicate a central role for non-histone protein methylation in pluripotency but a thorough characterization of proteins involved in this modification is lacking. To fill this gap, we aim to define a proteome-wide expression portrait of methyl-transferases and de-methylases present in these cells, to characterize their downstream substrates, including all possible methylation forms and to analyze the molecular rewiring caused by perturbation of key proteins involved in the control of this modification in pluripotent cells. First, we found that, compared to terminally differentiated cells, mESCs express significantly higher levels of most methyl-transferases and de-methylases encoded in the genome. Next, we performed a comprehensive characterization of the arginine and lysine protein methylomes in mESCs and murine fibroblasts, identifying ̴ 4,300 and 167 arginine and lysine methylation sites respectively. This analysis revealed hundreds of novel sites and methylated proteins, including important pluripotency factors. Finally, to deconvolute the complex relationships between these enzymes and their potential downstream methylated substrates, we carried out a thermal stability assay. This allowed us to identify system-wide changes in response to chemical inhibitors for three important methyl-transferases and de-methylases in pluripotent cells, thereby revealing new processes regulated by these enzymes. These biophysical analyses showed that Prmt5 is involved in multiple processes related to RNA biology including stress granules assembly. On the other hand, Ezh2 inhibition induces changes in the thermal stability of proteins involved cell cycle and mitotic chromosomes in mESCs. Finally, Kdm1a inhibition activates the epithelial-to-mesenchymal transition in mESCs via Zeb1-CoREST repressive complex. Collectively, our datasets represent a rich resource to study the role of protein methylation in the control of pluripotency and stemness. In addition, the thermal stability approach implemented here is applicable to analyze protein methylation functions in other biological contexts, such as cancer