Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsOur 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.
Highlights
The histone methyltransferase SETDB1 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
Distinct functional requirements of the SETDB1‐binding region and the fibronectin type-III (FNIII) domain in ATF7IP‐dependent retroelement silencing We have previously established Atf7ip KO cells using mouse embryonic stem cells (mESCs) infected with the murine stem cell virus (MSCV) carrying the GFP gene as a background [22] and observed that the Atf7ip KO ESCs showed increased expression of SETDB1-regulated ERVs and the MSCV-GFP reporter [16]
For rescue experiments with ATF7IP, we used a piggyBac transposase-based vector for the expression of 3xFLAG-tagged mouse ATF7IP with either WT or each domain’s deletion mutants: dSETDB1 lacking residues 627–694, which is within the corresponding SETDB1 binding domain of mouse ATF7IP and covering two estimated α-helix regions, and dFNIII lacking residues 1190–1306 of the FNIII domain, which is highly conserved between human and mouse ATF7IP (Fig. 1a and Additional file 1: Fig. S1)
Summary
The histone methyltransferase SETDB1 ( 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). Methylated histone H3 lysine 9 (H3K9) is associated with gene silencing, and it is dynamically regulated by several methyltransferases and demethylases. We have shown that in mESCs, one of the lysine methyltransferases, SETDB1 ( known as ESET) suppresses the expression of Class I and II endogenous retroviruses (ERVs) by depositing H3K9me marks [2, 3]. TRIM28 ( known as KAP1 or TIF1B) and TRIM28associated nucleic acid-binding zinc-finger proteins (ZFPs), including KRAB-ZFPs and YY1, play an important role in SETDB1 targeting to ERVs and silencing them [4,5,6]. Differences between the two silencing mechanisms have not yet been fully clarified
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