DNA methylation is an essential epigenetic mark in mammals that has to be re-established after each round of DNA replication. The protein UHRF1 is essential for this process; it has been proposed that the protein targets newly replicated DNA by cooperatively binding hemi-methylated DNA and H3K9me2/3, but this model leaves a number of questions unanswered. Here, we present evidence for a direct recruitment of UHRF1 by the replication machinery via DNA ligase 1 (LIG1). A histone H3K9-like mimic within LIG1 is methylated by G9a and GLP and, compared with H3K9me2/3, more avidly binds UHRF1. Interaction with methylated LIG1 promotes the recruitment of UHRF1 to DNA replication sites and is required for DNA methylation maintenance. These results further elucidate the function of UHRF1, identify a non-histone target of G9a and GLP, and provide an example of a histone mimic that coordinates DNA replication and DNA methylation maintenance.

Understanding of the appropriate regulation of enzymatic activities of histone‐modifying enzymes remains poor. The lysine methyltransferase, SETDB1, is one of the enzymes responsible for the methylation of histone H3 at lysine 9 (H3K9) and plays a key role in H3K9 trimethylation‐mediated silencing of genes and retrotransposons. Here, we reported that how SETDB1's enzymatic activities can be regulated by the nuclear protein, ATF7IP, a known binding partner of SETDB1. Mechanistically, ATF7IP mediates SETDB1 retention inside the nucleus, presumably by inhibiting its nuclear export by binding to the N‐terminal region of SETDB1, which harbors the nuclear export signal motifs, and also by promoting its nuclear import. The nuclear localization of SETDB1 increases its ubiquitinated, enzymatically more active form. Our results provided an insight as to how ATF7IP can regulate the histone methyltransferase activity of SETDB1 accompanied by its nuclear translocation.

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.Electronic supplementary materialThe online version of this article (10.1186/s13072-018-0231-z) contains supplementary material, which is available to authorized users.

Mdm2, a ubiquitin ligase and downstream effector of p53, attenuates the proliferative potential of primary cells via ubiquitination and degradation of the glycolytic enzyme PGAM under senescence-inducing stress.

Highlights d The crystal structure of UHRF1 TTD domain bound to the LIG1K126me3 was determined d Arg121 of LIG1 is a key residue for high-affinity binding to the TTD d Phosphorylation of LIG1T123 negatively regulates the interaction with UHRF1 d LIG1K126me3 binding changes UHRF1 structure from closed to open

At fertilization, the paternal genome undergoes extensive reprogramming through protamine-histone exchange and active DNA demethylation, but only a few maternal factors have been defined in these processes. We identified maternal Mettl23 as a protein arginine methyltransferase (PRMT), which most likely catalyzes the asymmetric dimethylation of histone H3R17 (H3R17me2a), as indicated by in vitro assays and treatment with TBBD, an H3R17 PRMT inhibitor. Maternal histone H3.3, which is essential for paternal nucleosomal assembly, is unable to be incorporated into the male pronucleus when it lacks R17me2a. Mettl23 interacts with Tet3, a 5mC-oxidizing enzyme responsible for active DNA demethylation, by binding to another maternal factor, GSE (gonad-specific expression). Depletion of Mettl23 from oocytes resulted in impaired accumulation of GSE, Tet3, and 5hmC in the male pronucleus, suggesting that Mettl23 may recruit GSE-Tet3 to chromatin. Our findings establish H3R17me2a and its catalyzing enzyme Mettl23 as key regulators of paternal genome reprogramming.

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Background The histone methyltransferase SETDB1 (also known as ESET) represses genes and various types of transposable elements, such as endogenous retroviruses (ERVs) and integrated exogenous retroviruses, through a deposition of trimethylation on lysine 9 of histone H3 (H3K9me3) in mouse embryonic stem cells (mESCs). ATF7IP (also known as MCAF1 or AM), a binding partner of SETDB1, regulates the nuclear localization and enzymatic activities of SETDB1 and plays a crucial role in SETDB1-mediated transcriptional silencing. In this study, we further dissected the ATF7IP function with its truncated mutants in Atf7ip knockout (KO) mESCs. Results We demonstrated that the SETDB1-interaction region within ATF7IP is essential for ATF7IP-dependent SETDB1 nuclear localization and silencing of both ERVs and integrated retroviral transgenes, whereas its C-terminal fibronectin type-III (FNIII) domain is dispensable for both these functions; rather, it has a role in efficient silencing mediated by the SETDB1 complex. Proteomic analysis identified a number of FNIII domain-interacting proteins, some of which have a consensus binding motif. We showed that one of the FNIII domain-binding proteins, ZMYM2, was involved in the efficient silencing of a transgene by ATF7IP. RNA-seq analysis of Atf7ip KO and WT or the FNIII domain mutant of ATF7IP-rescued Atf7ip KO mESCs showed that the FNIII domain mutant re-silenced most de-repressed SETDB1/ATF7IP-targeted ERVs compared to the WT. However, the silencing activity of the FNIII domain mutant was weaker than that of the ATF7IP WT, and some of the de-repressed germ cell-related genes in Atf7ip KO mESCs were not silenced by the FNIII domain mutant. Such germ cell-related genes are targeted and silenced by the MAX/MGA complex, and MGA was also identified as another potential binding molecule of the ATF7IP FNIII domain in the proteomic analysis. This suggests that the FNIII domain of ATF7IP acts as a binding hub of ATF7IP-interacting molecules possessing a specific interacting motif we named FAM and contributes to one layer of the SETDB1/ATF7IP complex-mediated silencing mechanisms. Conclusions Our findings contributed to further understanding the function of ATF7IP in the SETDB1 complex, revealed the role of the FNIII domain of ATF7IP in transcriptional silencing, and suggested a potential underlying molecular mechanism for it.