Tri-methylation of ATF7IP by G9a/GLP recruits the chromodomain protein MPP8

Abstract

Abstract Background G9a and the related enzyme GLP were originally identified as histone lysine methyltransferases and then shown to also methylate several other non-histone proteins. Results Here, we performed a comprehensive screen to identify their substrates in mouse embryonic stem cells (mESCs). We identified 59 proteins, including histones and other known substrates. One of the identified substrates, activating transcriptional factor 7-interacting protein 1 (ATF7IP), is tri-methylated at a histone H3 lysine 9 (H3K9)-like mimic by the G9a/GLP complex, although this complex mainly introduces di-methylation on H3K9 and DNA ligase 1 (LIG1) K126 in cells. The catalytic domain of G9a showed a higher affinity for di-methylated lysine on ATF7IP than LIG1, which may create different methylation levels of different substrates in cells. Furthermore, we found that M-phase phosphoprotein 8 (MPP8), known as a H3K9me3-binding protein, recognizes methylated ATF7IP via its chromodomain. MPP8 is also a known component of the human silencing hub complex that mediates silencing of transgenes via SETDB1 recruitment, which is a binding partner of ATF7IP. Although the interaction between ATF7IP and SETDB1 does not depend on ATF7IP methylation, we found that induction of SETDB1/MPP8-mediated reporter-provirus silencing is delayed in mESCs expressing only an un-methylatable mutant of ATF7IP. Conclusions Our findings provide new insights into the roles of lysine methylation in non-histone substrates which are targeted by the G9a/GLP complex and suggest a potential function of ATF7IP methylation in SETDB1/MPP8-mediated transgene silencing.

Keywords

• G9a/Ehmt2
• GLP/Ehmt1
• ATF7IP
• MPP8
• Non-histone methylation
• Transgene silencing