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Kinetic characterization of human histone H3 lysine 36 methyltransferases, ASH1L and SETD2.

Biochimica et biophysica acta (2015-05-24)
Mohammad S Eram, Ekaterina Kuznetsova, Fengling Li, Evelyne Lima-Fernandes, Steven Kennedy, Irene Chau, Cheryl H Arrowsmith, Matthieu Schapira, Masoud Vedadi
ABSTRACT

Dysregulation of methylation of lysine 36 on histone H3 (H3K36) have been implicated in a variety of diseases including cancers. ASH1L and SETD2 are two enzymes among others that catalyze H3K36 methylation. H3K4 methylation has also been reported for ASH1L. Radioactivity-based enzyme assays, Western and immunoblotting using specific antibodies and molecular modeling were used to characterize substrate specificity of ASH1L and SETD2. Here we report on the assay development and kinetic characterization of ASH1L and SETD2 and their substrate specificities in vitro. Both enzymes were active with recombinant nucleosome as substrate. However, SETD2 but not ASH1L methylated histone peptides as well indicating that the interaction of the basic post-SET extension with substrate may not be critical for SETD2 activity. Both enzymes were not active with nucleosome containing a H3K36A mutation indicating their specificity for H3K36. Analyzing the methylation state of the products of ASH1L and SETD2 reactions also confirmed that both enzymes mono- and dimethylate H3K36 and are inactive with H3K4 as substrate, and that only SETD2 is able to trimethylate H3K36 in vitro. We determined the kinetic parameters for ASH1L and SETD2 activity enabling screening for inhibitors that can be used to further investigate the roles of these two proteins in health and disease. Both ASH1L and SETD2 are H3K36 specific methyltransferases but only SETD2 can trimethylate this mark. The basic post-SET extension is critical for ASH1L but not SETD2 activity. We provide full kinetic characterization of ASH1L and SETD2 activity.

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