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.

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.

Histone H3 lysine 9 dimethylation (H3K9me2) is a highly conserved silencing epigenetic mark. Chromatin marked with H3K9me2 forms large domains in mammalian cells and overlaps well with lamina-associated domains and the B compartment defined by Hi-C. However, the role of H3K9me2 in 3-dimensional (3D) genome organization remains unclear. Here, we investigated genome-wide H3K9me2 distribution, transcriptome, and 3D genome organization in mouse embryonic stem cells following the inhibition or depletion of H3K9 methyltransferases (MTases): G9a, GLP, SETDB1, SUV39H1, and SUV39H2. We show that H3K9me2 is regulated by all five MTases; however, H3K9me2 and transcription in the A and B compartments are regulated by different MTases. H3K9me2 in the A compartments is primarily regulated by G9a/GLP and SETDB1, while H3K9me2 in the B compartments is regulated by all five MTases. Furthermore, decreased H3K9me2 correlates with changes to more active compartmental state that accompanied transcriptional activation. Thus, H3K9me2 contributes to inactive compartment setting.

Kleefstra syndrome (KS) (9q34 deletion syndrome) is a rare autosomal dominant disorder characterized by intellectual disability, frequently coupled with a spectrum of complex physical and clinical manifestations. As the euchromatic histone methyltransferase-1 gene (EHMT1, GLP, or KMT1D) within the 9q34 region is deleted or mutated in most of the individuals with KS, its absence or defect in one allele is speculated to cause the major symptoms of the syndrome. Most of the EHMT1 mutations are frameshift or nonsense mutations, but two individuals with KS were reported to possess EHMT1 missense mutations. These two mutations have been predicted to cause a defective enzymatic function, but precise biochemical validation was not conducted. Therefore, we validated these two mutations by performing in vitro histone methyltransferase (HMT) activity assay and found that C1073Y and R1197W mutations severely affected the HMT activity. Additionally, the same amino-acid substitutions in mouse GLP induced impairment of in vivo GLP function. Furthermore, these two EHMT1 mutants showed defective heterocomplex formation with G9a (partner HMT) which is essential for their in vivo HMT function. Conclusively, our biochemical characterization clearly demonstrates that the previously reported two missense mutations of EHMT1 deteriorate HMT activity and GLP function, which presumably cause KS.

Epigenetic information (epigenome) on chromatin is crucial for the determination of cellular identity and for the expression of cell type-specific biological functions. The cell type-specific epigenome is maintained beyond replication and cell division. Nucleosomes of chromatin just after DNA replication are a mixture of old histones with the parental epigenome and newly synthesized histones without such information. The diluted epigenome is mostly restored within one cell cycle using the epigenome on the parental DNA and nucleosomes as replication templates. However, many important questions about the epigenome replication process remain to be clarified. In this study, we investigated the model system comprising of dimethylated histone H3 lysine 9 (H3K9me2) and its regulation by the lysine methyltransferase G9a. Using this epigenome model system, we addressed whether H3K9me2 can be induced in specific cell cycle stages, especially G1. Using cell cycle-specific degrons, we achieved G1 or late G1-to M phases specific accumulation of exogenous G9a in G9a deficient cells. Importantly, global levels of H3K9me2 were significantly recovered by both cell types. These data indicate that H3K9me2 may be plastic and inducible, even in the long-living, terminally-differentiated, post-mitotic, G0-G1 cell population in vivo. This knowledge is valuable in designing epigenome-manipulation-based treatments for diseases.