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.
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机译：通过在小鼠胚胎干细胞中的组蛋白H3（H3K9ME3）的赖氨酸9上的赖氨酸9上的赖氨酸9上沉积，抑制基因和各种类型的转移元素，例如内源性逆转录病毒（ERV）和集成的外源逆转录病毒（麦塞斯）。 ATF7IP（也称为McAF1或AM），SetdB1的结合伴侣调节SetdB1的核定位和酶活性，并在SetDB1介导的转录沉默中起着至关重要的作用。在这项研究中，我们将ATF7IP在ATF7IP敲除（KO）MESCS中的截短突变体进行了解剖。我们证明ATF7IP内的SetDB1相互作用区域对于ATF7IP依赖的SETDB1核定位和沉默的核定位和钝化逆转录病毒转基因是必不可少的，而其C末端纤连蛋白-III（FNIII）结构域对于这两种功能可分配;相反，它在由SetDB1复合物中介导的有效沉默中具有作用。蛋白质组学分析鉴定了许多FNIII结构域相互作用的蛋白质，其中一些蛋白质具有共有结合基序。我们表明，其中一个FNIII结构域结合蛋白Zmym2涉及通过ATF7IP的转基因的有效沉默。 ATF7IP KO和WT的RNA-SEQ分析或ATF7IP-RESCOPS KO MESCS的FNIII结构域突变体表明，与WT相比，FNIII结构域突变体重新沉默最脱抑制的SETDB1 / ATF7IP靶向ERV。然而，FNIII结构域突变体的沉默活性弱于ATF7IP WT的活性，并且ATF7IP KO MESCS中的一些脱抑制的生殖细胞相关基因未被FNIII结构域突变体沉默。这些生殖细胞相关基因被最大/ MgA络合物靶向并沉默，并且MGA也被鉴定为蛋白质组学分析中ATF7IP FNIII结构域的另一个潜在结合分子。这表明ATF7IP的FNIII结构域作为ATF7IP - 相互作用分子的结合集线器，其具有特定的相互作用基序，其命名为FAM并有助于一层SETDB1 / ATF7IP复合介导的沉默机构。我们的研究结果有助于进一步了解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，并提出了它的潜在的潜在的分子机制。
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