Abstract
Clr4 is a histone H3 lysine 9 methyltransferase in Schizosaccharomyces pombe that is essential for heterochromatin formation. Previous biochemical and structural studies have shown that Clr4 is in an autoinhibited state in which an autoregulatory loop (ARL) blocks the active site. Automethylation of lysine residues in the ARL relieves autoinhibition. To investigate the mechanism of Clr4 regulation by autoinhibition and automethylation, we exchanged residues in the ARL by site-directed mutagenesis leading to stimulation or inhibition of automethylation and corresponding changes in Clr4 catalytic activity. Furthermore, we demonstrate that Clr4 prefers monomethylated (H3K9me1) over unmodified (H3K9me0) histone peptide substrates, similar to related human enzymes and, accordingly, H3K9me1 is more efficient in overcoming autoinhibition. Due to enzyme activation by automethylation, we observed a sigmoidal dependence of Clr4 activity on the AdoMet concentration, with stimulation at high AdoMet levels. In contrast, an automethylation-deficient mutant showed a hyperbolic Michaelis–Menten type relationship. These data suggest that automethylation of the ARL could act as a sensor for AdoMet levels in cells and regulate the generation and maintenance of heterochromatin accordingly. This process could connect epigenome modifications with the metabolic state of cells. As other human protein lysine methyltransferases (for example, PRC2) also use automethylation/autoinhibition mechanisms, our results may provide a model to describe their regulation as well.
Highlights
Post-translational modifications (PTMs) of proteins control many of their functional properties in a dynamic and reversible way [1,2]
Previous data have shown Clr4 in an autoinhibited state, because its autoregulatory loop (ARL) binds to the active site cleft and thereby blocks binding of methylation external substrates [40]
Due to the positioning of lysine residues next to the active center, automethylation of Clr4 can occur at residues K455 and K472 in the ARL
Summary
Post-translational modifications (PTMs) of proteins control many of their functional properties in a dynamic and reversible way [1,2]. Protein lysine methylation on histones and non-histone proteins is one of the most important and abundant PTMs that can impact regulatory and functional mechanisms of the corresponding proteins, linking the dysregulation of this modification to many diseases [2,4,5]. The fission yeast Schizosaccharomyces pombe (S. pombe) protein lysine methyltransferase (PKMT) Clr, a homolog of the Drosophila Su(var) and the human SUV39H1 and SUV39H2 enzymes, introduces methylation of lysine 9 on histone H3 [10,11]. It is the only active H3K9-specific PKMT in this species. A disordered region between the CD and SET domain binds to the nucleosome core contributing to Clr activity [16]
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