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.

ATF7IP regulates SETDB1 nuclear localization and increases its ubiquitination

“…Finally, our results could provide a potential explanation for the discrepancy between two previous studies on the regulation of methyltransferase activity of SETDB1 by ATF7IP [5,34]. In both these studies, SETDB1 that was produced in insect cells was used.…”

“…would be different. Thus, we speculated that the ubiquitination level of SETDB1 in the complex was higher in the ATF7IP coexpressed condition shown in the study by Wang et al [5], but not in the study conducted by Basavapathruni et al [34], and that the difference in the ubiquitination status between the two studies could be the reason for inconsistent results. In support of this idea, the addition of ATF7IP after the purification did not increase the enzymatic activity of SETDB1 in in vitro as shown in the study conducted by Wang et al [5].…”

“…In a pioneering study, it was proposed that ATF7IP facilitates SETDB1‐mediated conversion of H3K9me2 to H3K9me3 by an unknown mechanism . However, in another report, it was argued that ATF7IP does not enhance the catalytic activity of SETDB1 in in vitro . Another notion for the in vivo regulation of SETDB1 by ATF7IP in human cells is that it contributes to the stability of SETDB1 in the nucleus .…”

ATF7IP-Mediated Stabilization of the Histone Methyltransferase SETDB1 Is Essential for Heterochromatin Formation by the HUSH Complex

“…Initially, ATF7IP was proposed to act as a direct modulator of the catalytic activity of SETDB1, with mAM, the murine homolog of ATF7IP, shown to stimulate the catalytic activity of murine SETDB1 in vitro and promote the efficient conversion of di-methyl H3K9 to the tri-methylated state (Wang et al., 2003). However, a recent study found little effect of human ATF7IP on the in vitro catalytic activity of human SETDB1 (Basavapathruni et al., 2016), leading to the suggestion that the in vivo role of ATF7IP could be to regulate the spatial or temporal recruitment of SETDB1 to target sites on chromatin (Basavapathruni et al., 2016, Koch et al., 2009). …”

MET-2, a SETDB1 family methyltransferase, coordinates embryo events through distinct histone H3 methylation states

“…By sequence, MET-2 is most similar to vertebrate SETDB1 (Poulin et al 2005;Andersen and Horvitz 2007). Both enzymes can catalyze mono and di-methylation on H3K9, and directly or indirectly regulate H3K9me3 (Basavapathruni et al, 2016;Loyola et al, 2009;Towbin et al, 2012;Wang et al, 2003). SET domain methyltransferases contain a 'switch position' in their catalytic site that determines the degree of methylation, with bulkier residues able to accommodate mono-and di-but not tri-methylation (Jih et al, 2017).…”