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Updated: Feb 4, 2026

In vitro Methylation Assay to Study Protein Arginine Methylation
Published on: October 5, 2014
Tri-methylation of ATF7IP by G9a/GLP recruits the chromodomain protein MPP8
Takeshi Tsusaka1,2, Masaki Kikuchi3, Tadahiro Shimazu1
1Cellular Memory Laboratory, RIKEN Cluster for Pioneering Research, Wako, 351-0198, Japan.
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.
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