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ATF7IP regulates SETDB1 nuclear localization and increases its ubiquitination.

ABSTRACT: 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.

SUBMITTER: Tsusaka T

PROVIDER: S-EPMC6893292 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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ATF7IP regulates SETDB1 nuclear localization and increases its ubiquitination.

Tsusaka Takeshi T Shimura Chikako C Shinkai Yoichi Y

EMBO reports 20191002 12

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 media ...[more]

PMID: 31576654

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Project description:X chromosome inactivation (XCI) is a developmental program of heterochromatin formation that initiates during early female mammalian embryonic development and is maintained through a lifetime of cell divisions in somatic cells. Despite identification of the crucial long non-coding RNA Xist and involvement of specific chromatin modifiers in the establishment and maintenance of the heterochromatin of the inactive X chromosome (Xi), interference with known pathways only partially reactivates the Xi once silencing has been established. Here, we studied ATF7IP (MCAF1), a protein previously characterized to coordinate DNA methylation and histone H3K9 methylation through interactions with the methyl-DNA binding protein MBD1 and the histone H3K9 methyltransferase SETDB1, as a candidate maintenance factor of the Xi.We found that siRNA-mediated knockdown of Atf7ip in mouse embryonic fibroblasts (MEFs) induces the activation of silenced reporter genes on the Xi in a low number of cells. Additional inhibition of two pathways known to contribute to Xi maintenance, DNA methylation and Xist RNA coating of the X chromosome, strongly increased the number of cells expressing Xi-linked genes upon Atf7ip knockdown. Despite its functional importance in Xi maintenance, ATF7IP does not accumulate on the Xi in MEFs or differentiating mouse embryonic stem cells. However, we found that depletion of two known repressive biochemical interactors of ATF7IP, MBD1 and SETDB1, but not of other unrelated H3K9 methyltransferases, also induces the activation of an Xi-linked reporter in MEFs.Together, these data indicate that Atf7ip acts in a synergistic fashion with DNA methylation and Xist RNA to maintain the silent state of the Xi in somatic cells, and that Mbd1 and Setdb1, similar to Atf7ip, play a functional role in Xi silencing. We therefore propose that ATF7IP links DNA methylation on the Xi to SETDB1-mediated H3K9 trimethylation via its interaction with MBD1, and that this function is a crucial feature of the stable silencing of the Xi in female mammalian cells.

Project description: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.

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ATF7IP regulates SETDB1 nuclear localization and increases its ubiquitination.

Publications

ATF7IP regulates SETDB1 nuclear localization and increases its ubiquitination.

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