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. 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. 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.

中文翻译：

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ATF7IP的纤连蛋白III型（FNIII）域有助于SETDB1复合物介导的有效转录沉默

组蛋白甲基转移酶SETDB1（也称为ESET）通过在小鼠胚胎干细胞中组蛋白H3（H3K9me3）的赖氨酸9上沉积三甲基化来抑制基因和各种类型的转座因子，例如内源性逆转录病毒（ERV）和整合的外源性逆转录病毒。 （mESC）。SETB1的结合伙伴ATF7IP（也称为MCAF1或AM）调节SETDB1的核定位和酶活性，并在SETDB1介导的转录沉默中起关键作用。在这项研究中，我们进一步剖析了Atf7ip基因敲除（KO）mESCs中的ATF7IP功能及其截短的突变体。我们证明了ATF7IP中的SETDB1相互作用区域对于依赖ATF7IP的SETDB1核定位以及ERV和整合逆转录病毒转基因的沉默至关重要，而其C端纤连蛋白III型（FNIII）结构域对于这两种功能都是必不可少的；而是在SETDB1复合体介导的有效沉默中起作用。蛋白质组学分析鉴定了许多FNIII域相互作用蛋白，其中一些具有共有结合基序。我们表明，FNIII域结合蛋白之一ZMYM2，参与了ATF7IP对转基因的有效沉默。对Atf7ip KO和WT或ATF7IP拯救的Atf7ip KO mESC的FNIII结构域突变体的RNA序列分析表明，与WT相比，FNIII结构域突变体使大多数被抑制的SETDB1 / ATF7IP靶向ERV沉默。然而，FNIII结构域突变体的沉默活性比ATF7IP WT弱，并且Atf7ip KO mESCs中一些抑制的生殖细胞相关基因未被FNIII结构域突变体沉默。这种生殖细胞相关基因被MAX / MGA复合物靶向并沉默，并且在蛋白质组学分析中，MGA还被确定为ATF7IP FNIII结构域的另一个潜在结合分子。这表明ATF7IP的FNIII结构域充当ATF7IP相互作用分子的结合枢纽，该分子具有我们称为FAM的特定相互作用基序，并有助于SETDB1 / ATF7IP复合物介导的沉默机制的一层。我们的发现有助于进一步理解ATF7IP在SETDB1复合物中的功能，揭示了ATF7IP的FNIII结构域在转录沉默中的作用，并暗示了其潜在的潜在分子机制。在蛋白质组学分析中，MGA还被确定为ATF7IP FNIII结构域的另一个潜在结合分子。这表明ATF7IP的FNIII结构域充当ATF7IP相互作用分子的结合枢纽，该分子具有我们称为FAM的特定相互作用基序，并有助于SETDB1 / ATF7IP复合物介导的沉默机制的一层。我们的发现有助于进一步理解ATF7IP在SETDB1复合物中的功能，揭示了ATF7IP的FNIII域在转录沉默中的作用，并暗示了其潜在的潜在分子机制。在蛋白质组学分析中，MGA还被确定为ATF7IP FNIII结构域的另一个潜在结合分子。这表明ATF7IP的FNIII结构域充当ATF7IP相互作用分子的结合枢纽，该分子具有我们称为FAM的特定相互作用基序，并有助于SETDB1 / ATF7IP复合物介导的沉默机制的一层。我们的发现有助于进一步理解ATF7IP在SETDB1复合物中的功能，揭示了ATF7IP的FNIII结构域在转录沉默中的作用，并暗示了其潜在的潜在分子机制。