Abstract 104: ATF7IP does not alter the substrate specificity of the lysine methyltransferase SETDB1

The histone methyltransferase (HMT) 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. Its activity has been shown to be regulated by its partner protein ATF7IP, and here we examine the contribution of ATF7IP to the in vitro activity and substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed in Sf21 insect cells and 1:1 stoichiometric complexes were purified for comparison against apo-SETDB1 enzyme. We employed both radiometric flashplate-based and MALDI mass spectrometry assays to follow methylation on histone 3 15-mer peptides where lysine 9 was either unmodified, mono-, or di-methylated. These two methods provide orthogonal readouts on H3K9 methylation: the radiometric flashplate-based assay employs 3H-labeled SAM and provides quantitation of labeled, methylated peptide. The MALDI mass spectrometry assay offers quantitation of consumption and accumulation of each individual H3K9 methylation state (H3K9me0, me1, me2, and me3). Results show that both apo-SETDB1 and SETDB1:ATF7IP complex catalyzed both monomethylation and dimethylation of H3K9 peptide substrates, but were unable to perform H3K9 trimethylation. While ATF7IP did not impact the substrate methylation profile, the activity of the complex was lower than apo-SETDB1 by a factor of 4. This difference was due to a decrease in the value of kcat as the substrate KM values were comparable between apo-SETDB1 and the SETDB1:ATF7IP complex. ATF7IP therefore does not alter SETDB19s substrate specificity. H3K9 monomethylation and dimethylation by SETDB1 occurred in a distributive manner and 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 H3K9me1 and H3K9me2. The results presented here indicate that ATF7IP does not alter substrate specificity of SETDB1 in vitro, and though it decreases SETDB19s catalytic activity, our data points to a nonenzymatic function of ATF7IP in regulating SETDB1 function. Citation Format: Aravind Basavapathruni, Jodi Gureasko, Margaret Porter Scott, P. Ann Boriack-Sjodin, Timothy J. Wigle, Thomas V. Riera, Robert A. Copeland. ATF7IP does not alter the substrate specificity of the lysine methyltransferase SETDB1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 104. doi:10.1158/1538-7445.AM2015-104