Reference number : GC-2017-Q1-02136, amendment number : , agreement type : G, recipient legal name : Li, Shawn Shun-Cheng (The University of Western Ontario), recipient country : CA, recipient province : nan, recipient city : Ontario, recipient postal code : nan, program name english : Discovery Grants Program - Individual, program name french : Programme de subventions à la découverte - individuelles, program purpose english : Despite the importance of Lys methylation in normal physiology and the pathogenesis of complex diseases such as cancer and neurological disorder, we know little about this post-translational modification (PTM) beyond its role on histone modification. We propose here to define the enzyme-substrate network of lysine methyltransferases (KMTs) and methyllyinse demethylases (KDMs) in order to understand the function of Lys methylation. As an important step towards this ambitious goal, we will focus on characterizing the KMTs and KDMs that modify H3K4 methylation, a histone mark that is associated with actively transcribed genes. The importance of H3K4 methylation is underscored, on the one hand, by the large number of KMTs (&gt;12) and KDMs (&gt;6) that are capable of modifying the mark, and, on the other, by the lethality or severe developmental defects in mice in which the corresponding genes are ablated. We hypothesize that the H3K4-specific KMTs/KDMs are in fact non-specific; but rather, they possess activities towards additional histone and non-histone proteins . For this Discovery Grant, we will determine the substrate specificity and identify the methylation network for the H3K4-modifying KMTs and KDMs. Our Objectives are : 
 
 (1) We will determine the substrate specificity of the KMTs and KDMs by in vitro methylation/demethylation assays using peptide microarrays and complementary proteomics and bioinformatics approaches . The resulting specificity profile for a given KMT/KDM will allow us to predict the substrates for that enzyme. We will also use the peptide array approach to identify inhibitors that may be exploited as functional probes. 
 
 (2) We will elucidate the KTM/KDM-substrate network in cells by advanced mass spectrometry. To this end, we will tag a KTM/KDM, express it in cells and carry out affinity purification (AP)-tandem mass spectrometry (MS/MS) and AP-MRM (Multiple Reaction Monitoring) analysis to identify (by MS/MS) novel substrates or validate (by MRM) the above predicted KTM/KDM-substrate network. 
 
 (3) We will characterize the function of H3K4-modifying KMTs/KDMs in stem cell differentiation . Specifically, we will knock down or knock out individual KMTs/KDMs from mouse embryonic stem cells and characterize defects of the resulting clones in neural differentiation. 
 
Our work would not only uncover numerous novel Lys methylation sites and provide unprecedented insights into the specificity and function of modifying enzymes that converges on H3K4, the approach developed herein could be used for systematic identification and functional characterization of Lys methylation on a global scale. This, in turn, would enable the development of innovative strategies for disease intervention targeting Lys methylation., program purpose french : Despite the importance of Lys methylation in normal physiology and the pathogenesis of complex diseases such as cancer and neurological disorder, we know little about this post-translational modification (PTM) beyond its role on histone modification. We propose here to define the enzyme-substrate network of lysine methyltransferases (KMTs) and methyllyinse demethylases (KDMs) in order to understand the function of Lys methylation. As an important step towards this ambitious goal, we will focus on characterizing the KMTs and KDMs that modify H3K4 methylation, a histone mark that is associated with actively transcribed genes. The importance of H3K4 methylation is underscored, on the one hand, by the large number of KMTs (&gt;12) and KDMs (&gt;6) that are capable of modifying the mark, and, on the other, by the lethality or severe developmental defects in mice in which the corresponding genes are ablated. We hypothesize that the H3K4-specific KMTs/KDMs are in fact non-specific; but rather, they possess activities towards additional histone and non-histone proteins . For this Discovery Grant, we will determine the substrate specificity and identify the methylation network for the H3K4-modifying KMTs and KDMs. Our Objectives are : 
 
 (1) We will determine the substrate specificity of the KMTs and KDMs by in vitro methylation/demethylation assays using peptide microarrays and complementary proteomics and bioinformatics approaches . The resulting specificity profile for a given KMT/KDM will allow us to predict the substrates for that enzyme. We will also use the peptide array approach to identify inhibitors that may be exploited as functional probes. 
 
 (2) We will elucidate the KTM/KDM-substrate network in cells by advanced mass spectrometry. To this end, we will tag a KTM/KDM, express it in cells and carry out affinity purification (AP)-tandem mass spectrometry (MS/MS) and AP-MRM (Multiple Reaction Monitoring) analysis to identify (by MS/MS) novel substrates or validate (by MRM) the above predicted KTM/KDM-substrate network. 
 
 (3) We will characterize the function of H3K4-modifying KMTs/KDMs in stem cell differentiation . Specifically, we will knock down or knock out individual KMTs/KDMs from mouse embryonic stem cells and characterize defects of the resulting clones in neural differentiation. 
 
Our work would not only uncover numerous novel Lys methylation sites and provide unprecedented insights into the specificity and function of modifying enzymes that converges on H3K4, the approach developed herein could be used for systematic identification and functional characterization of Lys methylation on a global scale. This, in turn, would enable the development of innovative strategies for disease intervention targeting Lys methylation., agreement title english : Systematic characterization of the enzyme-substrate network in protein Lysine methylation, agreement title french : Systematic characterization of the enzyme-substrate network in protein Lysine methylation, agreement number : RGPIN, agreement value : 170000.0, agreement start date : 2017-05-10, agreement end date : nan, description english : nan, description french : nan, expected results english : nan, expected results french : nan, additional information english : Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023), additional information french : Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023), owner organization : nserc-crsng, owner organization title : Natural Sciences and Engineering Research Council of Canada | Conseil de recherches en sciences naturelles et en génie du Canada