title: Characterization of the Enzymatic Activity of SETDB1 and Its 1:1 Complex with ATF7IP.
abstract: The protein methyltransferase (PMT) SETDB1 is a strong candidate oncogene in melanoma and lung carcinomas. SETDB1 methylates lysine 9 of histone 3 (H3K9), utilizing S-adenosylmethionine (SAM) as the methyl donor and its catalytic activity, has been reported to be regulated by a partner protein ATF7IP. Here, we examine the contribution of ATF7IP to the in vitro activity and substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed and 1:1 stoichiometric complexes were purified for comparison against SETDB1 enzyme alone. We employed both radiometric flashplate-based and SAMDI mass spectrometry assays to follow methylation on histone H3 15-mer peptides, where lysine 9 was either unmodified, monomethylated, or dimethylated. Results show that SETDB1 and the SETDB1:ATF7IP complex efficiently catalyze both monomethylation and dimethylation of H3K9 peptide substrates. The activity of the binary complex was 4-fold lower than SETDB1 alone. This difference was due to a decrease in the value of kcat as the substrate KM values were comparable between SETDB1 and the SETDB1:ATF7IP complex. H3K9 methylation by SETDB1 occurred in a distributive manner, and this too was unaffected by the presence of ATF7IP. This finding is important as H3K9 can be methylated by HMTs other than SETDB1 and a distributive mechanism would allow for interplay between multiple HMTs on H3K9. Our results indicate that ATF7IP does not directly modulate SETDB1 catalytic activity, suggesting alternate roles, such as affecting cellular localization or mediating interaction with additional binding partners.
authors.affiliation: Epizyme, Inc., 400 Technology Square, Cambridge, Massachusetts 02139, United States.
authors.forename: Aravind
authors.initials: A
authors.lastname: Basavapathruni
authors.affiliation: Epizyme, Inc., 400 Technology Square, Cambridge, Massachusetts 02139, United States.
authors.forename: Jodi
authors.initials: J
authors.lastname: Gureasko
authors.affiliation: Epizyme, Inc., 400 Technology Square, Cambridge, Massachusetts 02139, United States.
authors.forename: Margaret
authors.initials: M
authors.lastname: Porter Scott
authors.affiliation: Blue Sky BioServices, Worcester, Massachusetts 01605, United States.
authors.forename: William
authors.initials: W
authors.lastname: Hermans
authors.affiliation: Aldevron, Madison, Wisconsin 53719, United States.
authors.forename: Adarsh
authors.initials: A
authors.lastname: Godbole
authors.affiliation: Aldevron, Madison, Wisconsin 53719, United States.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Leland
authors.affiliation: Epizyme, Inc., 400 Technology Square, Cambridge, Massachusetts 02139, United States.
authors.forename: P Ann
authors.initials: PA
authors.lastname: Boriack-Sjodin
authors.affiliation: Epizyme, Inc., 400 Technology Square, Cambridge, Massachusetts 02139, United States.
authors.forename: Tim J
authors.initials: TJ
authors.lastname: Wigle
authors.affiliation: Epizyme, Inc., 400 Technology Square, Cambridge, Massachusetts 02139, United States.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Copeland
authors.affiliation: Epizyme, Inc., 400 Technology Square, Cambridge, Massachusetts 02139, United States.
authors.forename: Thomas V
authors.initials: TV
authors.lastname: Riera
mesh_terms: D011495:Histone-Lysine N-Methyltransferase; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008745:Methylation; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D011496:Protein Methyltransferases / Q000737:chemistry* / Q000378:metabolism; D012097:Repressor Proteins; D012436:S-Adenosylmethionine / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
chemical_list: C501309:ATF7IP protein, human; D006657:Histones; D046912:Multiprotein Complexes; D012097:Repressor Proteins; D014157:Transcription Factors; D012436:S-Adenosylmethionine; D011496:Protein Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; C454062:SETDB1 protein, human