Abstract
BackgroundG9a and the related enzyme GLP were originally identified as histone lysine methyltransferases and then shown to also methylate several other non-histone proteins.ResultsHere, 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.ConclusionsOur 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.
Highlights
G9a and the related enzyme GLP were originally identified as histone lysine methyltransferases and shown to methylate several other non-histone proteins
We further examined the endogenous interaction of activating transcriptional factor 7-interacting protein 1 (ATF7IP) with M-phase phosphoprotein 8 (MPP8) using the chromatin fraction of mouse embryonic stem cells (mESCs) (Fig. 5h)
Our screen succeeded in the identification of known and previously unknown G9a/GLP targets in mESCs
Summary
G9a and the related enzyme GLP were originally identified as histone lysine methyltransferases and shown to methylate several other non-histone proteins. G9a and GLP are SET-domain-type lysine methyltransferases that primarily exist as a heteromeric complex in vivo [11] Recent work has established that the G9a/ GLP-mediated non-histone methylations are involved in gene expression and the maintenance of DNA methylation during DNA replication, independently from H3K9 methylation [17,18,19]. These findings let us reconsider the G9a/GLP roles yet to be distinguished as either H3K9 methylation-dependent or methylationindependent. A more comprehensive screen of non-histone G9a/GLP substrates would be worthful
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
